Reagents for directed biomarker signal amplification

ABSTRACT

Described herein are methods, compositions and articles of manufacture involving neutral conjugated polymers including methods for synthesis of neutral conjugated water-soluble polymers with linkers along the polymer main chain structure and terminal end capping units. Such polymers may serve in the fabrication of novel optoelectronic devices and in the development of highly efficient biosensors. The invention further relates to the application of these polymers in assay methods.

CROSS-REFERENCE

This application is a continuation of application Ser. No. 15/239,713,filed Aug. 17, 2016 now U.S. patent Ser. No. 10/094,838; whichapplication is a continuation of application Ser. No. 14/821,386, filedAug. 7, 2015 and issued as U.S. Pat. No. 9,547,008; which application isa continuation of application Ser. No. 14/018,985, filed Sep. 5, 2013and issued as U.S. Pat. No. 9,139,869; which application is acontinuation of application Ser. No. 13/009,764, filed Jan. 19, 2011 andissued as U.S. Pat. No. 8,575,303, which claims the benefit of U.S.Provisional Application Ser. No. 61/296,379, filed Jan. 19, 2010 andU.S. Provisional Application Ser. No. 61/358,406, filed Jun. 24, 2010,which applications are incorporated herein by reference in theirentireties.

BACKGROUND OF THE INVENTION

Fluorescent hybridization probes have developed into an important toolin the sequence-specific detection of DNA and RNA. The signals generatedby the appended fluorescent labels (or dyes) can be monitored in realtime and provide simple, rapid, and robust methods for the detection ofbiological targets and events. Utility has been seen in applicationsranging from microarrays and real time PCR to fluorescence in situhybridization (FISH).

Recent work in the area of multichromophores, particularly regardingconjugated polymers (CPs) has highlighted the potential these materialshave in significantly improving the detection sensitivity of suchmethods (Liu and Bazan, Chem. Mater., 2004). The light harvestingstructures of these materials can be made water soluble and adapted toamplify the fluorescent output of various probe labels (See U.S. patentapplication Ser. No. 10/600,286, filed Jun. 20, 2003 and Gaylord,Heeger, and Bazan, Proc. Natl. Acad. Sci., 2002, both of which areincorporated herein by reference in their entirety).

Results such as these indicate CPs to be highly promising in the fieldof nucleic acid diagnostics, particularly where sample quantities arescarce. However, there exist methods for the amplification (orreplication) of nucleic acid targets, i.e., PCR. Comparatively, in thefield of protein recognition, there are no such simple methods foramplifying the targeted materials. As such, signal enhancement arisingfrom CP application is of high consequence in this area.

Dye-labeled antibodies are regularly used for the detection of proteintargets in applications such as immunohistochemistry, protein arrays,ELISA tests, and flow cytometry. Integrating CP materials into suchmethodologies promises to provide a dramatic boost in the performance ofsuch assays, enabling detection levels previously unattainable withconventional fluorescent reporters (e.g., dyes).

Beyond addition signal, one of the other key drivers in biologicaldetection formats is the ability to detect multiple analytes in the sametest or multiplexing. This is commonly achieved by using fluorescentreporters with operate at different, discernable wavelengths. CPmaterials are ideally suited to provide a platform for expandedmultiplexing. This can be achieved by tuning the structure of differentCPs to operate at different wavelengths or by incorporating a dye withinthe polymer-biomolecule conjugate.

The material and methods to produce higher sensitivity biological assaysand increase multiplexing are highly desired in both molecular (nucleicacid) and immunoassay formats.

SUMMARY OF THE INVENTION

Provided herein are water soluble conjugated polymers of Formula (I):

wherein:

each R is independently a non-ionic side group capable of impartingsolubility in water in excess of 10 mg/mL;

MU is a polymer modifying unit or band gap modifying unit that is evenlyor randomly distributed along the polymer main chain and is optionallysubstituted with one or more optionally substituted substituentsselected from halogen, hydroxyl, C₁-C₁₂ alkyl, C₂-C₁₂ alkene, C₂-C₁₂alkyne, C₃-C₁₂ cycloalkyl, C₁-C₁₂ haloalkyl, C₁-C₁₂ alkoxy,C₂-C₁₈(hetero)aryloxy, C₂-C₁₈(hetero)arylamino,(CH₂)_(x′)(OCH₂CH₂)_(y′)OCH₃ where each x′ is independently an integerfrom 0-20, y′ is independently an integer from 0 to 50, or aC₂-C₁₈(hetero)aryl group;

each optional linker L₁ and L₂ are aryl or heteroaryl groups evenly orrandomly distributed along the polymer main chain and are substitutedwith one or more pendant chains terminated with a functional group forconjugation to another substrate, molecule or biomolecule selected fromamine, carbamate, carboxylic acid, carboxylate, maleimide, activatedesters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones,azide, alkyne, aldehydes, thiols, and protected groups thereof;

G₁ and G₂ are each independently selected from hydrogen, halogen, amine,carbamate, carboxylic acid, maleimide, activated esters,N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azide,alkyne, aldehydes, thiol, optionally substituted aryl, optionallysubstituted heteroaryl, halogen substituted aryl, boronic acidsubstituted aryl, boronic ester substituted aryl, boronic esters,boronic acids, optionally substituted fluorene and aryl or heteroarylsubstituted with one or more pendant chains terminated with a functionalgroup selected from amine, carbamate, carboxylic acid, carboxylate,maleimide, activated esters, N-hydroxysuccinimidyl, hydrazines,hydrazides, hydrazones, azide, alkyne, aldehydes, thiols, and protectedgroups thereof for conjugation to another substrate, molecule orbiomolecule;

wherein the polymer comprises at least 1 functional group selected fromamine, carbamate, carboxylic acid, carboxylate, maleimide, activatedesters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones,azide, alkyne, aldehydes, and thiols within G₁, G₂, L₁ or L₂ thatallows, for functional conjugation to another molecule, substrate orbiomolecule;

each dashed bond, - - - - - - , is independently a single bond, triplebond or optionally substituted vinylene (—CR⁵═CR⁵—) wherein each R⁵ isindependently hydrogen, cyano, C₁-C₁₂ alkyl, C₂-C₁₂ alkene, C₂-C₁₂alkyne, C₃-C₁₂ cycloalkyl or a C₂-C₁₈(hetero)aryl group, wherein eachC₁-C₁₂ alkyl, C₂-C₁₂ alkene, C₂-C₁₂ alkyne, C₃-C₁₂ cycloalkyl or aC₂-C₁₈(hetero)aryl group is optionally substituted with one or morehalogen, hydroxyl, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl group,C₁-C₁₂ alkoxy, or C₁-C₁₂ haloalkyl; and

n is an integer from 1 to about 10,000; and

a, b, c and d define the mol % of each unit within the structure whicheach can be evenly or randomly repeated and where a is a mol % from 10to 100%, b is a mol % from 0 to 90%, and each c and d are mol % from 0to 25%.

In one aspect, water soluble conjugated polymers of Formula (I) have thestructure of Formula (Ia):

wherein R, L₁, L₂, G₁, G₂, MU, a, b, c, d and n are described previouslyfor formula (I).

In some embodiments, each R is independently (CH₂)_(x)(OCH₂CH₂)_(y)OCH₃where each x is independently an integer from 0-20, each y isindependently an integer from 0 to 50, or a benzyl optionallysubstituted with one or more halogen, hydroxyl, C₁-C₁₂ alkoxy, or(OCH₂CH₂)_(z)OCH₃ where each z is independently an integer from 0 to 50.In some instances, each R is (CH₂)₃(OCH₂CH₂)₁₁OCH₃.

In other embodiments, each R is a benzyl substituted with at least one(OCH₂CH₂)₁₀OCH₃ group. In some instances, the benzyl is substituted withtwo (OCH₂CH₂)₁₀OCH₃ groups. In other instances, the benzyl issubstituted with three (OCH₂CH₂)₁₀OCH₃ groups.

In some embodiments, optional linkers L₁ or L₂ have the structure:

*=Site for covalent attachment to unsaturated backbone; wherein R³ isindependently hydrogen, halogen, alkoxy(C₁-C₁₂), C₁-C₁₂ alkyl, C₂-C₁₂alkene, C₂-C₁₂ alkyne, C₃-C₁₂ cycloalkyl or a C₂-C₁₈(hetero)aryl group,wherein each C₁-C₁₂ alkyl, C₂-C₁₂ alkene, C₂-C₁₂ alkyne, C₃-C₁₂cycloalkyl or a C₂-C₁₈(hetero)aryl group is optionally substituted withone or more halogen, hydroxyl, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂alkynyl group, C₁-C₁₂ alkoxy, or C₁-C₁₂ haloalkyl; and q is an integerfrom 0 to 4.

In other embodiments, optional linkers L₁ or L₂ have the structure:

*=site for covalent attachment to unsaturated backbone wherein A is asite for conjugation, chain extension or crosslinking and is—[O—CH₂—CH₂]_(q)—W, or (C₁-C₁₂)alkoxy-X or C₂-C₁₈(hetero)aryl, phenoxy,amido, amino, carbamate, carboxylate, carbonates, sulfide, disulfide, orimido groups terminated with a functional group selected from amine,carbamate, carboxylate, carboxylic acid, maleimide, activated esters,N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azide,alkyne, aldehydes, thiols, and protected groups thereof for conjugationto another substrate, molecule or biomolecule; W is —OH or —COOH; X is—NH₂, —NHCOOH, —NHCOOC(CH₃)₃,—NHCO(C₃-C₁₂)cycloalkyl(C₁-C₄)alkyl-N-maleimide; or—NHCO[CH₂—CH₂—O]_(t)NH₂; q is an integer from 1 to 20; and t is aninteger from 1 to 8.

In yet other embodiments, optional linkers L₁ or L₂ have the structure:

*=site for covalent attachment to backbone

wherein R²⁵ are each independently any one of or a combination of abond, C₁-C₂₀ alkyl, C₁-C₂₀ alkoxy, C₂-C₂₀ alkene, C₂-C₂₀ alkyne, C₃-C₂₀cycloalkyl, C₁-C₂₀ haloalkyl, (CH₂)_(x)(OCH₂CH₂)_(p)(CH₂)_(x) where eachx is independently an integer from 0-20, p is independently an integerfrom 0 to 50, aryl, C₂-C₁₈(hetero)aryl, phenoxy, amido, amino,carbamate, carboxylate, carbonates, sulfide, disulfide, or imido groups;

wherein at least one R²⁵ is terminated with a functional group selectedfrom amine, carbamate, carboxylate, carboxylic acid, maleimide,activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides,hydrazones, azide, alkyne, aldehydes, thiols, and protected groupsthereof for conjugation to another substrate, molecule or biomolecule.

In further embodiments, optional linkers L₁ or L₂ have the structure:

*=site for covalent attachment to unsaturated backbone;

-   -   wherein R35 is any one of or a combination of a bond, C1-C20        alkyl, C1-C20 alkoxy, C2-C20 alkene, C2-C20 alkyne, C3-C20        cycloalkyl, C1-C20 haloalkyl, (CH2)x(OCH2CH2)p(CH2)x where each        x is independently an integer from 0-20, p is independently an        integer from 0 to 50, aryl, C2-C18(hetero)aryl, phenoxy, amido,        amino, carbamate, carboxylate, carbonates, sulfide, disulfide,        or imido groups terminated with a functional group selected from        amine, carbamate, carboxylate, carboxylic acid, maleimide,        activated esters, N-hydroxysuccinimidyl, hydrazines, hydrazides,        hydrazones, azide, alkyne, aldehydes, thiols, and protected        groups thereof for conjugation to another substrate, molecule or        biomolecule.

In further embodiments, optional linkers L₁ or L₂ are selected from thegroup consisting of a-h having the structures:

*=site for covalent attachment to unsaturated backbone;

wherein R′ is independently H, halogen, C₁-C₁₂ alkyl, (C₁-C₁₂ alkyl)NH₂,C₂-C₁₂ alkene, C₂-C₁₂ alkyne, C₃-C₁₂ cycloalkyl, C₁-C₁₂ haloalkyl,C₂-C₁₈(hetero)aryl, C₂-C₁₈(hetero)arylamino, —[CH₂—CH₂]r′—Z¹, or(C₁-C₁₂)alkoxy-X¹; and wherein Z¹ is —OH or —COOH; X¹ is —NH₂, —NHCOOH,—NHCOOC(CH₃)₃, —NHCO(C3-C12)cycloalkyl(C1-C4)alkyl-N-maleimide; or—NHCO[CH₂—CH₂—O]_(s′)(CH₂)_(s′)NH₂; r′ is an integer from 1 to 20; andeach s′ is independently an integer from 1 to 20,(CH₂)₃(OCH₂CH₂)_(x″)OCH₃ where x″ is independently an integer from 0 to50, or a benzyl optionally substituted with one or more halogen,hydroxyl, C₁-C₁₂ alkoxy, or (OCH₂CH₂)_(y″)OCH₃ where each y″ isindependently an integer from 0 to 50 and R′ is different from R;

wherein k is 2, 4, 8, 12 or 24;

wherein R¹⁵ is selected from the group consisting of l-t having thestructure:

*=site for covalent attachment to backbone.

In yet further embodiments, optional linkers L₁ or L₂ are

In some embodiments, G₁ and G₂ are each independently selected fromhydrogen, halogen, alkyne, optionally substituted aryl, optionallysubstituted heteroaryl, halogen substituted aryl, boronic acidsubstituted aryl, boronic ester substituted aryl, boronic esters,boronic acids, optionally substituted fluorine and aryl or heteroarylsubstituted with one or more pendant chains terminated with a functionalgroup, molecule or biomolecule selected from amine, carbamate,carboxylic acid, carboxylate, maleimide, activated esters,N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azide,alkyne, aldehydes, thiols, and protected groups thereof for conjugationto another substrate, molecule or biomolecule.

In some embodiments, G₁ and G₂ each independently have the structure

wherein R¹¹ is any one of or a combination of a bond, C₁-C₂₀ alkyl,C₁-C₂₀ alkoxy, C₂-C₂₀ alkene, C₂-C₂₀ alkyne, C₃-C₂₀ cycloalkyl, C₁-C₂₀haloalkyl, (CH₂)_(x)(OCH₂CH₂)_(p)(CH₂)_(x) where each x is independentlyan integer from 0-20, p is independently an integer from 0 to 50, aryl,C₂-C₁₈(hetero)aryl, phenoxy, amido, amino, carbamate, carboxylate,carbonates, sulfide, disulfide, or imido groups terminated with afunctional group selected from amine, carbamate, carboxylate, carboxylicacid, maleimide, activated esters, N-hydroxysuccinimidyl, hydrazines,hydrazides, hydrazones, azide, alkyne, aldehydes, thiols, and protectedgroups thereof for conjugation to another substrate, molecule orbiomolecule.

In other embodiments, G₁ and G₂ are each independently selected from thegroup consisting of 1-31 having the structures:

*=site for covalent attachment to backbonewherein R¹⁵ is selected from the group consisting of l-t having thestructure:

and k is 2, 4, 8, 12 or 24.

In further embodiments, G₁ and G₂ are optionally substituted aryl orheteroaryl wherein the optional substituent is selected from halogen,amine, carbamate, carboxylic acid, maleimide, activated esters,N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azide,alkyne, aldehydes, thiol, boronic acid, boronate radical, boronic estersand optionally substituted fluorene.

In some embodiments, G₁ and G₂ are the same. In other embodiments, G₁and G₂ are different. In further embodiments, the polymer contains asingle conjugation site at only one terminus of the polymer chain G₁ orG₂.

In yet further embodiments, G₁ and G₂ is

In some embodiments, MU is selected from the group consisting of a′-k′having the structure:

*=site for covalent attachment to unsaturated backbone

wherein R is a non-ionic side group capable of imparting solubility inwater in excess of 10 mg/mL.

In some embodiments, the water soluble conjugated polymer has thestructure of formula:

wherein at least one of G₁ or G₂ comprises a functional conjugationsite.

In some embodiments, the water soluble conjugated polymer has thestructure of formula:

wherein L₁ comprises a functional conjugation site.

In some embodiments, the water soluble conjugated polymer has thestructure of formula:

wherein at least one of G₁ or G₂ comprises a functional conjugationsite.

In other embodiments, the polymer has the structure of formula:

In other embodiments, the polymer has the structure of formula:

In other embodiments, the polymer has the structure of formula:

In other embodiments, the polymer has the structure of formula:

In other embodiments, the polymer has the structure of formula:

In other embodiments, the polymer has the structure of formula:

In other embodiments, the polymer has the structure of formula:

In other embodiments, the polymer has the structure of formula:

In some instances, a signaling chromophore is attached to the polymervia the NH₂ group. In certain instances, the signaling chromophore isCy3 or Dylight 594 dye. In certain instances, the linker,

is about 10% of the entire polymer. In other instances, the polymer isconjugated to a secondary dye reporter and an antibody.

In some embodiments of conjugated polymers described herein, the polymeris further conjugated to additional molecules. In some embodiments, thepolymer is conjugated to a streptavidin, antibody or nucleic acid andused as a direct fluorescent reporter. In certain embodiments, thepolymer is conjugated to a streptavidin. In other embodiments, thepolymer is conjugated to thiol groups at the hinge region of anantibody. In yet other embodiments, the polymer is conjugated to anamine group on a protein which is modified with a heterobifunctionallinker. In further embodiments, the polymer is conjugated to a nucleicacid. In yet further embodiments, the polymer is conjugated to anantibody. In certain instances, the polymer is conjugated to amonoclonal antibody, a secondary antibody or a primary antibody. Inother instances, a polymer antibody conjugate is excited at about 405 nmin a flow cytometry assay where the specific signal is at least 3 foldgreater than the same antibody conjugated to Pacific Blue.

In some embodiments of conjugated polymers described herein, the polymeris purified by ion exchange chromatography. In other embodiments, thepolymer is ≥95% pure.

In some embodiments of conjugated polymers described herein, the polymeris used in flow cytometry assays to identify different cell markers orcell types. In other embodiments, the polymer is used to sort cells. Inyet other embodiments, the polymer is used to sort cells for use intherapeutics.

In some embodiments of conjugated polymers described herein, the polymeris used for intracellular staining. In certain instances, the polymer isused in flow cytometry assays to identify different cell markers or celltypes.

In some embodiments of conjugated polymers described herein, the polymercomprises a minimum number average molecular weight of greater than40,000 g/mol and a water solubility of greater than 50 mg/mL in purewater or a phosphate buffered saline solution.

In some embodiments of conjugated polymers described herein, the polymercomprises at least two unique conjugation linkers which can conjugatedto two unique materials.

Also provided herein are assay methods comprising providing a samplethat is suspected of containing a target biomolecule; providing a sensorprotein conjugated to at least one signaling chromophore and is capableof interacting with the target biomolecule or a target-associatedbiomolecule; providing a water soluble conjugated polymer describedherein; contacting the sample with the sensor protein and the conjugatedpolymer in a solution under conditions in which the sensor protein canbind to the target biomolecule or a target-associated biomolecule ifpresent; applying a light source to the sample that can excite theconjugated polymer; and detecting whether light is emitted from thesignaling chromophore.

In some embodiments, the sensor protein is an antibody. In otherembodiments, the sensor protein comprises a plurality of sensor proteinsconjugated to a plurality of signaling chromophores, wherein at leasttwo of the plurality of chromophores emit different wavelengths of lightupon energy transfer from the multichromophore.

Also provided herein are conjugated polymer complexes comprising apolymer coupled to at least one biomolecule selected from the groupconsisting of a sensor biomolecule, a bioconjugate and a targetbiomolecule wherein the polymer is covalently bound by at least onebioconjugation site pendant thereto, and the polymer comprises asignaling chromophore or a signaling chromophore optionally iscovalently bound to the polymer or the sensor biomolecule; wherein thepolymer comprises the structure of formula:

wherein:

each R is a non-ionic side group capable of imparting solubility inwater in excess of 10 mg/mL;

MU is a polymer modifying unit or band gap modifying unit that is evenlyor randomly distributed along the polymer main chain and is optionallysubstituted with one or more optionally substituted substituentsselected from halogen, hydroxyl, C₁-C₁₂ alkyl, C₂-C₁₂ alkene, C₂-C₁₂alkyne, C₃-C₁₂ cycloalkyl, C₁-C₁₂ haloalkyl, C₁-C₁₂ alkoxy,C₂-C₁₈(hetero)aryloxy, C₂-C₁₈(hetero)arylamino,(CH₂)_(x′)(OCH₂CH₂)_(y′)OCH₃ where each x′ is independently an integerfrom 0-20, y′ is independently an integer from 0 to 50, or aC₂-C₁₈(hetero)aryl group;

each optional linker L₁ and L₂ are aryl or heteroaryl groups evenly orrandomly distributed along the polymer main chain and are substitutedwith one or more pendant chains terminated with a functional group forconjugation to another molecule, substrate or biomolecule selected fromamine, carbamate, carboxylic acid, carboxylate, maleimide, activatedesters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones,azide, alkyne, aldehydes, thiols, and protected groups thereof;

G₁ and G₂ are each independently selected from hydrogen, halogen, amine,carbamate, carboxylic acid, maleimide, activated esters,N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azide,alkyne, aldehydes, thiol, optionally substituted aryl, optionallysubstituted heteroaryl, halogen substituted aryl, boronic acidsubstituted aryl, boronic ester substituted aryl, boronic esters,boronic acids, optionally substituted fluorene and aryl or heteroarylsubstituted with one or more pendant chains terminated with a functionalgroup selected from amine, carbamate, carboxylic acid, carboxylate,maleimide, activated esters, N-hydroxysuccinimidyl, hydrazines,hydrazides, hydrazones, azide, alkyne, aldehydes, thiols, and protectedgroups thereof for conjugation to another substrate, molecule orbiomolecule;

wherein the polymer comprises at least 1 functional group selected fromamine, carbamate, carboxylic acid, carboxylate, maleimide, activatedesters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones,azide, alkyne, aldehydes, and thiols within G₁, G₂, L₁ or L₂ thatallows, for functional conjugation to another molecule, substrate orbiomolecule;

n is an integer from 1 to about 10,000; and a, b, c and d define the mol% of each unit within the structure which each can be evenly or

randomly repeated and where a is a mol % from 10 to 100%, b is a mol %from 0 to 90%,

and each c and d are mol % from 0 to 25%.

In some embodiments, the sensor biomolecule is selected from the groupconsisting of an avidin, streptavidin, neutravidin, avidinDN, andavidinD. In other embodiments, the conjugated polymer complex is furtherconfigured to bind to a complex selected from the group consisting of abiotin-labeled antibody, biotin-labeled protein, and biotin-labeledtarget biomolecule.

In further embodiments, the sensor biomolecule is an antibody. In yetfurther embodiments, both the signaling chromophore and the sensorbiomolecule are covalently linked to the multichromophore through aplurality of linkers. In some other embodiments, both the signalingchromophore and the sensor biomolecule are covalently linked to thepolymer through a central linking site that covalently binds thepolymer, the signaling chromophore and the sensor biomolecule. In yetother embodiments, the signaling chromophore, when covalently bound tothe polymer or the sensor biomolecule, is an organic dye.

Also provided herein are water soluble conjugated polymer having thestructure of Formula (Ia):

wherein:

each R is independently (CH₂)_(x)(OCH₂CH₂)_(y)OCH₃ where each x isindependently an integer from 0-20, y is independently an integer from 0to 50, or a benzyl optionally substituted with one or more halogen,hydroxyl, C₁-C₁₂ alkoxy, or (OCH₂CH₂)_(z)OCH₃ where each z isindependently an integer from 0 to 50;

each optional linker L₁ or L₂ is selected from the group consisting ofa-i having the structure

*=site for covalent attachment to unsaturated backbone

wherein R′ is independently H, halogen, C₁-C₁₂ alkyl, (C₁-C₁₂ alkyl)NH₂,C₂-C₁₂ alkene, C₂-C₁₂ alkyne, C₃-C₁₂ cycloalkyl, C₁-C₁₂ haloalkyl,C₂-C₁₈(hetero)aryl, C₂-C₁₈(hetero)arylamino, —[CH₂—CH₂]_(r′)—Z¹, or(C₁-C₁₂)alkoxy-X¹; and wherein Z¹ is —OH or —COOH; X¹ is —NH₂, —NHCOOH,—NHCOOC(CH₃)₃, —NHCO(C3-C12)cycloalkyl(C1-C4)alkyl-N-maleimide; or—NHCO[CH₂—CH₂—O]_(s′)(CH₂)_(s′)NH₂; r′ is an integer from 1 to 20; andeach s′ is independently an integer from 1 to 20,(CH₂)₃(OCH₂CH₂)_(x″)OCH₃ where x″ is independently an integer from 0 to50, or a benzyl optionally substituted with one or more halogen,hydroxyl, C₁-C₁₂ alkoxy, or (OCH₂CH₂)_(y″)OCH₃ where each y″ isindependently an integer from 0 to 50 and R′ is different from R;

wherein R¹⁵ is selected from the group consisting of l-t having thestructure:

-   -   and k is 2, 4, 8, 12 or 24;

*=site for covalent attachment to backbone

MU is a polymer modifying unit or band gap modifying unit that isselected from the group consisting of a′-k′ having the structure:

*=site for covalent attachment to unsaturated backbone;

wherein R is a non-ionic side group capable of imparting solubility inwater in excess of 10 mg/mL;

G₁ and G₂ are each independently selected from the group consisting of1-31 having the structures:

wherein the polymer comprises at least 1 functional group selected fromamine, carbamate, carboxylic acid, carboxylate, maleimide, activatedesters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones,azide, alkyne, aldehydes, and thiols within G₁, G₂, L₁ or L₂ thatallows, for functional conjugation to another molecule, substrate orbiomolecule;

n is an integer from 1 to about 10,000; and

a, b, c and d define the mol % of each unit within the structure whicheach can be evenly or

randomly repeated and where a is a mol % from 10 to 100%, b is a mol %from 0 to 90%,

and each c and d are mol % from 0 to 25%.

Also provided herein are water soluble conjugated polymer having thestructure of Formula:

wherein Ar is an aryl or heteroaryl and is optionally substituted withone or more optionally substituted substituents selected from halogen,hydroxyl, C₁-C₁₂ alkyl, C₂-C₁₂ alkene, C₂-C₁₂ alkyne, C₃-C₁₂ cycloalkyl,C₁-C₁₂ haloalkyl, C₁-C₁₂ alkoxy, C₂-C₁₈(hetero)aryloxy,C₂-C₁₈(hetero)arylamino, (CH₂)_(x′)(OCH₂CH₂)_(y′)OCH₃ where each x′ isindependently an integer from 0-20, y′ is independently an integer from0 to 50; and dashed bonds, L₁, L₂, G₁, G₂, MU, a, b, c, d and n aredescribed previously for formula (I).

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1. Schematic of binding of a conjugated polymer in one embodimentof the invention.

FIG. 2. Schematic of a bioconjugated polymer of one embodiment of theinvention.

FIG. 3. Schematic of exemplary conjugated polymers conjugated (A)antibody; (B) an avidin; (C) nucleic acid; (D) dye, e.g., chromophore.

FIG. 4. Schematic of (A) a polymer conjugated to dye-labeled antibodyresulting in FRET; (B) a polymer conjugated dye-labeled strepavidinresulting in FRET; (C) nucleic acid probe sequences labeled with aquencher molecule conjugated to a conjugated polymer; (D) nucleic acidprobe sequences labeled with a quencher molecule conjugated polymer-dyetandem complex.

FIG. 5. Schematic of various methods of assaying for a targetbiomolecule or target associated biomolecule. (A) Conjugated polymerlinked to a bioconjugate; (B) polymer and dye labeled antibodiesrecognize a common target; (C) sensor biomolecule conjugated to both adye and a second bioconjugate; (D) second bioconjugate and the signalingchromopohre both conjugated to a nucleic acid.

FIG. 6. Schematic of an addition of a second linking site within thepolymer.

FIG. 7. Schematic of a polymer conjugated to a dye and a biomolecule andresulting energy transfer (A) polymer is conjugated to both abioconjugate; (B) polymer is conjugated to a strepavidin and a dye; (C)polymer is conjugated to a nucleic acid and a dye.

FIG. 8. Schematic of indirect associations with a sensor biomolecule ortarget associated biomolecule. (A) biotinylated antibody interactingwith a covalent conjugate of the conjugated polymer; (B) biotinylatedantibody conjugated polymer-dye tandem complex; (C) biotinylated nucleicacid interacting with a covalent conjugate of the conjugated polymer;(D) biotinylated nucleic conjugated polymer-dye tandem complex; (E)nucleic acid with digoxygenin moiety interacting with a covalentconjugate of the conjugated polymer; (F) nucleic acid with digoxygeninmoiety conjugated polymer-dye tandem complex.

FIG. 9. Schematic of exemplary conjugated polymers conjugated tosecondary antibodies (A) and primary antibodies (B).

FIG. 10. Schematic of a sandwich-type complex. (A) conjugated polymercomplex bioconjugated to a strepavidin; (B) biotin-labeled 1° antibody eused to probe the target protein directly.

FIG. 11. Schematic of appending one or two phenyl capping units to afluorene polymer.

FIG. 12. Block diagram showing a representative example logic device.

FIG. 13. Block diagram showing a representative example of a kit.

FIG. 14. Schematic of a streptavidin conjugation with a conjugatedpolymer and the resulting conjugate structure (top) and Coomassiestained agarose gel representative of the streptavidin-attached CP(below).

FIG. 15. Representative acrylamide gel depiction of biotinylated polymeralone or bound to Cy5-labeled streptavidin.

FIG. 16. Schematic of streptavidin-attached conjugated polymer of FIG.14 binding to biotinylated microspheres (top) and plot of fluorescenceexcitation of control biotinylated microspheres and microspheres boundto streptavidin conjugated polymer.

FIG. 17. Schematic of streptavidin-attached conjugated polymer of FIG.14 selectively bound to biotinylated microspheres and energy transfer todye acceptors on co-localized streptavidin-dye conjugates (top) and plotof energy transfer from streptavidin-attached conjugated polymer to dyeacceptor (bottom).

FIG. 18. Schematic of biotinylated polymer of FIG. 14 binding tostreptavidin coated microspheres (top) and plot of fluorescenceexcitation of control streptavidin coated microspheres and microspheresbound to biotinylated polymer.

FIG. 19. Schematic of biotinylated polymer of FIG. 14 binding todye-labeled streptavidin conjugates and FRET (top); plot of energytransfer from biotinylated polymer to two different dye acceptors(bottom left) and titration plot of polymer saturation (bottom right).

FIG. 20. Flow cytometry analysis of CD4 marking of Cyto-trol cells with440 nm polymer-streptavidin-conjugates.

FIG. 21. (A) Polymer structure of Example 38b conjugated to (from leftto right) FITC, Cy3, DyLight 594 and DyLight633; (B) Comparison of thefluorescence of the dye (DyLight594) excited near its absorbance maximum(lower curve) and polymer-dye conjugate excited at 405 nm (upper curve);(C) Comparison of the fluorescent signal of the base polymer (no dye,peak emission near 420 nm) to that of the polymer-dye conjugate (peakemission near 620 nm).

FIG. 22. Plot of flow testing of monoclonal antibody (antiCD4)conjugates on whole lysed blood samples.

FIG. 23. Plot of florescence of a dye (DyLight594) and a polymer-dyeconjugate by excitation of dye at 594 nm and the polymer-dye conjugateat 380 nm.

FIG. 24. Plot of fluorescent immunoassay (ELISA) withstreptavidin-attached conjugated polymer.

FIG. 25. Plot of fluorescent intensity vs. temperature of a DNAoligomer-polymer conjugate hybridized to a target.

FIG. 26. Ion exchange chromatogram for a polymer antibody conjugate toremove free polymer (left) and an SEC chromatogram showing theseparation of final conjugate from free antibody. In both chromatogramsabsorbance was monitored at 280 nm (lower curves) and 407 nm (uppercurves).

FIG. 27. Sandwich immunoassay on Luminex assay (left) and correspondingresults on the Luminex system using 532 nm excitation of both theconjugated polymer and PE streptavidin detection conjugates.

FIG. 28. Data on left show results obtained with compensation beadswhile the data set on the right results from a 4 color assay on humanblood samples.

FIGS. 29. (A) and (B) Schematic of covalent linkage of conjugatedpolymer to 2° antibody.

FIG. 30. Schematic of conjugated polymers in Fluorescent Immuno Assay(FIA). (A) conjugated polymer covalently linked to a detection antibody;(B) biotin binding protein covalently bound to the conjugated polymerand interacting with a biotinylated detection antibody; (C) secondaryantibody covalently linked to the conjugated polymer and interactingwith a detection antibody.

FIG. 31. (A) Schematic of nucleic acid probe sequences labeled with aquencher molecule conjugated to a conjugated polymer; (B) nucleic acidprobe sequences labeled with a quencher molecule conjugated to aconjugated polymer-dye tandem complex.

FIG. 32. Schematic of modifications of the HybProbe detection technique.(A) conjugated polymer covalently linked to the donor probe andresulting energy transfer to acceptor probe; (B) “Signal off”modification of the HybProbe approach where the conjugated polymer isquenched by an acceptor probe.

FIG. 33. Comparison of non-specific binding in various polymers (top) ina Jurkat cell (lymphocyte cell line) model; (bottom) plot ranking thepolymers in terms of signal generated purely by non-specific binding(NSB).

FIG. 34. Histograms collected from flow cytometry analysis (405 nmexcitation in a BD LSR-II cytometer) using a Jurkat cell line; (left)unstained cells and a negative control, anionic P4 polymer; (middle)range of different polymer and polymer side chain combinations tested onthe same cells; (right) neutral polymer P20 showed almost no off setfrom the untreated cells.

FIG. 35. Gel electrophoresis depicting relative mobility of avidin as afunction of the degree of conjugation with polymer AA1.

FIG. 36. Fractionation of crude polymer-avidin conjugate mixtures on aSuperdex 200 size exclusion column; (top) monitoring fractions by UVabsorbance; (bottom) gel electrophoresis of selected fractions tovisualize the degree to which avidin was attached to polymer.

FIG. 37. Gel electrophoresis of conjugation reactions performed withpolymer in varying molar excess to streptavidin; (left) UV illumination;(right) 532 nm excitation.

FIG. 38. Plot depicting purification of polymer streptavidin conjugateswith polymers exemplified in Example 9, denoted P30, (top) crudesamples; (bottom) purified conjugates).

FIG. 39. Enrichment of linker-functionalized polymers.

FIG. 40. Conjugation of amine polymer to streptavidin protein.

FIG. 41. Synthesis of a substituted divinylbenzene polymer and Endlinker incorporation.

FIG. 42. Conjugation of polymer to an amine on a primary antibody.

FIG. 43. Conjugation of polymer to an antibody using maleimide/thiolchemistry and partially reduced antibodies.

FIG. 44. Malemide/thiol conjugation of polymers to thiol modifiedantibodies.

FIG. 45. Azide polymer conjugation to alkyne terminated DNA oligomer.

FIG. 46. Polymer tandem conjugation to a primary antibody usingmaleimide/thiol chemistry.

DETAILED DESCRIPTION OF THE INVENTION

Before the present invention is described in further detail, it is to beunderstood that this invention is not limited to the particularmethodology, devices, solutions or apparatuses described, as suchmethods, devices, solutions or apparatuses can, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention.

Use of the singular forms “a,” “an,” and “the” include plural referencesunless the context clearly dictates otherwise. Thus, for example,reference to “an aggregation sensor” includes a plurality of aggregationsensors, reference to “a probe” includes a plurality of probes, and thelike. Additionally, use of specific plural references, such as “two,”“three,” etc., read on larger numbers of the same subject less thecontext clearly dictates otherwise.

Terms such as “connected,” “attached,” “conjugated” and “linked” areused interchangeably herein and encompass direct as well as indirectconnection, attachment, linkage or conjugation unless the contextclearly dictates otherwise; in one example, the phrase “conjugatedpolymer” is used in accordance with its ordinary meaning in the art andrefers to a polymer containing an extended series of unsaturated bonds,and that context dictates that the term “conjugated” should beinterpreted as something more than simply a direct or indirectconnection, attachment or linkage.

Where a range of values is recited, it is to be understood that eachintervening integer value, and each fraction thereof, between therecited upper and lower limits of that range is also specificallydisclosed, along with each subrange between such values. The upper andlower limits of any range can independently be included in or excludedfrom the range, and each range where either, neither or both limits areincluded is also encompassed within the invention. Where a value beingdiscussed has inherent limits, for example where a component can bepresent at a concentration of from 0 to 100%, or where the pH of anaqueous solution can range from 1 to 14, those inherent limits arespecifically disclosed. Where a value is explicitly recited, it is to beunderstood that values which are about the same quantity or amount asthe recited value are also within the scope of the invention, as areranges based thereon. Where a combination is disclosed, eachsubcombination of the elements of that combination is also specificallydisclosed and is within the scope of the invention. Conversely, wheredifferent elements or groups of elements are disclosed, combinationsthereof are also disclosed. Where any element of an invention isdisclosed as having a plurality of alternatives, examples of thatinvention in which each alternative is excluded singly or in anycombination with the other alternatives are also hereby disclosed; morethan one element of an invention can have such exclusions, and allcombinations of elements having such exclusions are hereby disclosed.

Unless defined otherwise or the context clearly dictates otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. Although any methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the invention, the preferred methods and materials are nowdescribed.

All publications mentioned herein are hereby incorporated by referencefor the purpose of disclosing and describing the particular materialsand methodologies for which the reference was cited. The publicationsdiscussed herein are provided solely for their disclosure prior to thefiling date of the present application. Nothing herein is to beconstrued as an admission that the invention is not entitled to antedatesuch disclosure by virtue of prior invention.

Definitions

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

“Alkyl” refers to a branched, unbranched or cyclic saturated hydrocarbongroup of 1 to 24 carbon atoms optionally substituted at one or morepositions, and includes polycyclic compounds. Examples of alkyl groupsinclude optionally substituted methyl, ethyl, n-propyl, isopropyl,n-butyl, s-butyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl,n-heptyl, n-octyl, n-decyl, hexyloctyl, tetradecyl, hexadecyl, eicosyl,tetracosyl and the like, as well as cycloalkyl groups such ascyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, adamantyl, and norbornyl. The term “lower alkyl” refers toan alkyl group of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms.Exemplary substituents on substituted alkyl groups include hydroxyl,cyano, alkoxy, ═O, ═S, —NO₂, halogen, haloalkyl, heteroalkyl,carboxyalkyl, amine, amide, thioether and —SH.

“Alkoxy” refers to an “—Oalkyl” group, where alkyl is as defined above.A “lower alkoxy” group intends an alkoxy group containing one to six,more preferably one to four, carbon atoms.

“Alkenyl” refers to a branched, unbranched or cyclic hydrocarbon groupof 2 to 24 carbon atoms containing at least one carbon-carbon doublebond optionally substituted at one or more positions. Examples ofalkenyl groups include ethenyl, 1-propenyl, 2-propenyl (allyl),1-methylvinyl, cyclopropenyl, 1-butenyl, 2-butenyl, isobutenyl,1,4-butadienyl, cyclobutenyl, 1-methylbut-2-enyl, 2-methylbut-2-en-4-yl,prenyl, pent-1-enyl, pent-3-enyl, 1,1-dimethylallyl, cyclopentenyl,hex-2-enyl, 1-methyl-1-ethylallyl, cyclohexenyl, heptenyl,cycloheptenyl, octenyl, cyclooctenyl, decenyl, tetradecenyl,hexadecenyl, eicosenyl, tetracosenyl and the like. Preferred alkenylgroups herein contain 2 to 12 carbon atoms. The term “lower alkenyl”intends an alkenyl group of 2 to 6 carbon atoms, preferably 2 to 4carbon atoms. The term “cycloalkenyl” intends a cyclic alkenyl group of3 to 8, preferably 5 or 6, carbon atoms. Exemplary substituents onsubstituted alkenyl groups include hydroxyl, cyano, alkoxy, ═O, ═S,—NO₂, halogen, haloalkyl, heteroalkyl, amine, thioether and —SH.

“Alkenyloxy” refers to an “—Oalkenyl” group, wherein alkenyl is asdefined above.

“Alkylaryl” refers to an alkyl group that is covalently joined to anaryl group.

Preferably, the alkyl is a lower alkyl. Exemplary alkylaryl groupsinclude benzyl, phenethyl, phenopropyl, 1-benzylethyl, phenobutyl,2-benzylpropyl and the like.

“Alkylaryloxy” refers to an “—Oalkylaryl” group, where alkylaryl is asdefined above.

“Alkynyl” refers to a branched or unbranched hydrocarbon group of 2 to24 carbon atoms containing at least one —C

“Amide” refers to —C(O)NR′R″, where R′ and R″ are independently selectedfrom hydrogen, alkyl, aryl, and alkylaryl.

“Amine” refers to an —N(R′)R″ group, where R′ and R″ are independentlyselected from hydrogen, alkyl, aryl, and alkylaryl.

“Aryl” refers to an aromatic group that has at least one ring having aconjugated pi electron system and includes carbocyclic, heterocyclic,bridged and/or polycyclic aryl groups, and can be optionally substitutedat one or more positions. Typical aryl groups contain 1 to 5 aromaticrings, which may be fused and/or linked. Exemplary aryl groups includephenyl, furanyl, azolyl, thiofuranyl, pyridyl, pyrimidyl, pyrazinyl,triazinyl, biphenyl, indenyl, benzofuranyl, indolyl, naphthyl,quinolinyl, isoquinolinyl, quinazolinyl, pyridopyridinyl,pyrrolopyridinyl, purinyl, tetralinyl and the like. Exemplarysubstituents on optionally substituted aryl groups include alkyl,alkoxy, alkylcarboxy, alkenyl, alkenyloxy, alkenylcarboxy, aryl,aryloxy, alkylaryl, alkylaryloxy, fused saturated or unsaturatedoptionally substituted rings, halogen, haloalkyl, heteroalkyl, —S(O)R,sulfonyl, —SO₃R, —SR, —NO₂, —NRR′, —OH, —CN, —C(O)R, —OC(O)R, —NHC(O)R,—(CH₂)_(n)CO₂R or —(CH₂)_(n)CONRR′ where n is 0-4, and wherein R and R′are independently H, alkyl, aryl or alkylaryl.

“Aryloxy” refers to an “—Oaryl” group, where aryl is as defined above.

“Carbocyclic” refers to an optionally substituted compound containing atleast one ring and wherein all ring atoms are carbon, and can besaturated or unsaturated.

“Carbocyclic aryl” refers to an optionally substituted aryl groupwherein the ring atoms are carbon.

“Halo” or “halogen” refers to fluoro, chloro, bromo or iodo. “Halide”refers to the anionic form of the halogens.

“Haloalkyl” refers to an alkyl group substituted at one or morepositions with a halogen, and includes alkyl groups substituted withonly one type of halogen atom as well as alkyl groups substituted with amixture of different types of halogen atoms. Exemplary haloalkyl groupsinclude trihalomethyl groups, for example trifluoromemyl.

“Heteroalkyl” refers to an alkyl group wherein one or more carbon atomsand associated hydrogen atom(s) are replaced by an optionallysubstituted heteroatom, and includes alkyl groups substituted with onlyone type of heteroatom as well as alkyl groups substituted with amixture of different types of heteroatoms. Heteroatoms include oxygen,sulfur, and nitrogen. As used herein, nitrogen heteroatoms and sulfurheteroatoms include any oxidized form of nitrogen and sulfur, and anyform of nitrogen having four covalent bonds including protonated forms.An optionally substituted heteroatom refers to replacement of one ormore hydrogens attached to a nitrogen atom with alkyl, aryl, alkylarylor hydroxyl.

“Heterocyclic” refers to a compound containing at least one saturated orunsaturated ring having at least one heteroatom and optionallysubstituted at one or more positions. Typical heterocyclic groupscontain 1 to 5 rings, which may be fused and/or linked, where the ringseach contain five or six atoms. Examples of heterocyclic groups includepiperidinyl, morpholinyl and pyrrolidinyl. Exemplary substituents foroptionally substituted heterocyclic groups are as for alkyl and aryl atring carbons and as for heteroalkyl at heteroatoms.

“Heterocyclic aryl” refers to an aryl group having at least 1 heteroatomin at least one aromatic ring. Exemplary heterocyclic aryl groupsinclude furanyl, thienyl, pyridyl, pyridazinyl, pyrrolyl, N-loweralkyl-pyrrolo, pyrimidyl, pyrazinyl, triazinyl, tetrazinyl, triazolyl,tetrazolyl, imidazolyl, bipyridyl, tripyridyl, tetrapyridyl, phenazinyl,phenanthrolinyl, purinyl, perylene, perylene diimide,diketopyrrolopyrrole, benzothiadiazol, benzoxadiazol, thienopyrazine andthe like.

“Hydrocarbyl” refers to hydrocarbyl substituents containing 1 to about20 carbon atoms, including branched, unbranched and cyclic species aswell as saturated and unsaturated species, for example alkyl groups,alkylidenyl groups, alkenyl groups, alkylaryl groups, aryl groups, andthe like. The term “lower hydrocarbyl” intends a hydrocarbyl group ofone to six carbon atoms, preferably one to four carbon atoms.

A “substituent” refers to a group that replaces one or more hydrogensattached to a carbon or nitrogen. Exemplary substituents include alkyl,alkylidenyl, alkylcarboxy, alkoxy, alkenyl, alkenylcarboxy, alkenyloxy,aryl, aryloxy, alkylaryl, alkylaryloxy, —OH, amide, carboxamide,carboxy, sulfonyl, ═O, ═S, —NO₂, halogen, haloalkyl, fused saturated orunsaturated optionally substituted rings, —S(O)R, —SO₃R, —SR, —NRR′,—OH, —CN, —C(O)R, —OC(O)R, —NHC(O)R, —(CH₂)_(n)CO₂R or —(CH₂)_(n)CONRR′where n is 0-4, and wherein R and R′ are independently H, alkyl, aryl oralkylaryl. Substituents also include replacement of a carbon atom andone or more associated hydrogen atoms with an optionally substitutedheteroatom.

“Sulfonyl” refers to —S(O)₂R, where R is alkyl, aryl, —C(CN)═C-aryl,—CH₂CN, alkylaryl, or amine.

“Thioamide” refers to —C(S)NR′R″, where R′ and R″ are independentlyselected from hydrogen, alkyl, aryl, and alkylaryl.

“Thioether” refers to —SR, where R is alkyl, aryl, or alkylaryl.

As used herein, the term “binding pair” refers to first and secondmolecules that bind specifically to each other with greater affinitythan to other components in the sample. The binding between the membersof the binding pair is typically noncovalent. Exemplary binding pairsinclude immunological binding pairs (e.g. any haptenic or antigeniccompound in combination with a corresponding antibody or binding portionor fragment thereof, for example digoxigenin and anti-digoxigenin,fluorescein and anti-fluorescein, dinitrophenol and anti-dinitrophenol,bromodeoxyuridine and anti-bromodeoxyuridine, mouse immunoglobulin andgoat anti-mouse immunoglobulin) and nonimmunological binding pairs(e.g., biotin-avidin, biotin-streptavidin, hormone [e.g., thyroxine andcortisol]-hormone binding protein, receptor-receptor agonist orantagonist (e.g., acetylcholine receptor-acetylcholine or an analogthereof) IgG-protein A, lectin-carbohydrate, enzyme-enzyme cofactor,enzyme-enzyme-inhibitor, and complementary polynucleotide pairs capableof forming nucleic acid duplexes) and the like. One or both member ofthe binding pair can be conjugated to additional molecules.

The terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and“nucleic acid molecule” are used interchangeably herein to refer to apolymeric form of nucleotides of any length, and may compriseribonucleotides, deoxyribonucleotides, analogs thereof, or mixturesthereof.

These terms refer only to the primary structure of the molecule. Thus,the terms includes triple-, double- and single-stranded deoxyribonucleicacid (“DNA”), as well as triple-, double- and single-strandedribonucleic acid (“RNA”). It also includes modified, for example byalkylation, and/or by capping, and unmodified forms of thepolynucleotide. Additional details for these terms as well as fordetails of base pair formation can be found in U.S. application Ser. No.11/344,942, filed Jan. 31, 2006, which is incorporate herein byreference in its entirety.

“Complementary” or “substantially complementary” refers to the abilityto hybridize or base pair between nucleotides or nucleic acids, such as,for instance, between a sensor peptide nucleic acid and a targetpolynucleotide. Complementary nucleotides are, generally, A and T (or Aand U), or C and G. Two single-stranded polynucleotides or PNAs are saidto be substantially complementary when the bases of one strand,optimally aligned and compared and with appropriate insertions ordeletions, pair with at least about 80% of the bases of the otherstrand, usually at least about 90% to 95%, and more preferably fromabout 98 to 100%.

Alternatively, substantial complementarity exists when a polynucleotideor PNA will hybridize under selective hybridization conditions to itscomplement. Typically, selective hybridization will occur when there isat least about 65% complementary over a stretch of at least 14 to 25bases, preferably at least about 75%, more preferably at least about 90%complementary. See, M. Kanehisa Nucleic Acids Res. 12:203(1984).

“Preferential binding” or “preferential hybridization” refers to theincreased propensity of one polynucleotide or PNA to bind to itscomplement in a sample as compared to a noncomplementary polymer in thesample.

Hybridization conditions for polynucleotides will typically include saltconcentrations of less than about 1M, more usually less than about 500mM and preferably less than about 200 mM. In the case of hybridizationbetween a peptide nucleic acid and a polynucleotide, the hybridizationcan be done in solutions containing little or no salt. Hybridizationtemperatures can be as low as 5° C., but are typically greater than 22°C., more typically greater than about 30° C., and preferably in excessof about 37° C. Longer fragments may require higher hybridizationtemperatures for specific hybridization. Other factors may affect thestringency of hybridization, including base composition and length ofthe complementary strands, presence of organic solvents and extent ofbase mismatching, and the combination of parameters used is moreimportant than the absolute measure of any one alone. Otherhybridization conditions which may be controlled include buffer type andconcentration, solution pH, presence and concentration of blockingreagents to decrease background binding such as repeat sequences orblocking protein solutions, detergent type(s) and concentrations,molecules such as polymers which increase the relative concentration ofthe polynucleotides, metal ion(s) and their concentration(s),chelator(s) and their concentrations, and other conditions known in theart.

“Multiplexing” herein refers to an assay or other analytical method inwhich multiple analytes can be assayed simultaneously.

“Having” is an open ended phrase like “comprising” and “including,” andincludes circumstances where additional elements are included andcircumstances where they are not.

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where the event or circumstance occurs and instances in whichit does not.

The embodiments disclosed herein relate generally to compositions ofconjugated polymer materials that contain active functional groups forconjugation (or attachment) to other molecules, substrates or the like.Certain embodiments describe methods and compositions that provide forspecific control of the incorporation and subsequent conjugation of suchfunctional sites. Linkers can be incorporated at one or both ends of aconjugated polymer chain or internally controlled by ratio of monomersused in the polymerizations. Such linkers can be the same or differentto allow for more than one distinct entity to be attached to theconjugated polymer structure.

Further embodiments describe conjugated polymer compositions that notonly provide for active conjugation sites but also are solubilizedthrough the use of non-ionic side chains (no formal charges). Suchembodiments exhibit exceptional water solubility and provide minimalinteractions with biological molecules and other common biological assaycomponents.

The embodiments disclosed herein further relate generally to assays andcomplexes including conjugated polymers useful for the identification oftarget biomolecules or biomolecules associated with target moleculesthrough enhanced signal afforded by their unique properties.

In certain general embodiments the conjugated polymer serves directly asthe optical reporter bound to a biomolecule, substrate or other assaycomponent. The conjugated polymers act as extended light harvestingstructures that when excited can absorb more energy than conventionalorganic dyes. The polymer then re-emits the light which can be detectedor measured. The signals generated from such conjugated polymercomplexes can be significantly greater than those obtained from otherfluorescent reporters.

In other embodiments one aspect includes energy transfer from conjugatedpolymers to dyes bound to the polymer or to a sensor which can be abiomolecule including a bioconjugate (e.g., an antibody, a streptavidinor nucleic acid sequence). In such embodiments it is common to observeamplified dye signal (relative to direct dye excitation) as a result ofthe conjugated polymer excitation and subsequent energy transfer.Further it is possible to use a range of dyes with varying energy tocreate a basis for a multicolor or multiplex detection format.

In certain embodiments the neutral conjugated polymers are bound toantibodies for the identification of specific cell markers and celltypes in flow cytometry and cell sorting assays. In other embodimentsthe conjugated polymers are further bound to a secondary dye reporter.In further embodiments the polymer and polymer-dye structures are boundto monoclonal antibodies.

In other embodiments the neutral conjugated polymers are bound toantibodies for use in various sandwich immunoassays.

In one embodiment, an approach modifying a format as followed inrelation to nucleic acid sensor assays as described in Gaylord, Heeger,and Bazan, J. Am. Chem. Soc., 2003 can be followed. Specifically, signalamplification of conjugated polymers can be based on binding events toindicate a hybridization event. Any established conjugated polymers canbe chosen as the donor, and one or more dye, preferably a dye with ahistory of efficient energy transfer, for example, fluorescein and Cy3,can be chosen as the acceptors. It is envisioned that the dye can bedirectly conjugated to a sensor molecule. As shown schematically in FIG.1, the sensor can be a biomolecule (e.g., an antibody) in a solution oron a substrate, to which conjugated polymers can be added. In theembodiment shown in FIG. 1, a dye can be covalently linked(bioconjugated) to an antibody (Y-shaped structure), which possesses anet negative charge. Addition of conjugated polymers (shown as wavylines) can result in interaction or binding between the conjugatedpolymer and the antibody, bringing the conjugated polymers and dye intoclose proximity. Interaction or binding can be achieved by any knownmethod including, but not limited to, avidin/biotin labeling. Distancerequirements for fluorescence resonance energy transfer (FRET) can thusbe met, and excitation of the polymer with light (shown as hν) resultsin amplified dye emission. It is envisioned that the conjugated polymerscan be excited at a wavelength where the dye does not have significantabsorbance. In one embodiment the dye emission can be at a longerwavelength than the conjugated polymer emission. In use it is envisionedthat an assay method can include the steps of providing a sample that issuspected of containing a target biomolecule, providing a sensorconjugated to a signaling chromophore and capable of interacting withthe target biomolecule, providing a conjugated polymer that interactswith the sensor and upon excitation is capable of transferring energy tothe sensor signaling chromophore and contacting the sample with thesensor and the conjugated polymer in a solution under conditions inwhich the sensor can bind to the target biomolecule if present. Next,the method can include applying a light source to the sample that canexcite the conjugated polymer, and detecting whether light is emittedfrom the signaling chromophore.

As disclosed herein, interaction or binding between conjugated polymersand dye-labeled antibodies can be a viable approach for increasingdetection sensitivities, for example of a biomolecule target. In afurther embodiment, covalently attaching the conjugated polymers to adye, biomolecule (e.g., an antibody complex) or both offers severaladvantages including reduced background and/or improved energy transfer.In the case of direct linkage to a biomolecule, biorecognition events,rather than non-specific polymer interaction or binding events (such asthose described above in FIG. 1), should govern conjugated polymerpresence. In this manner, nonspecific binding of conjugated polymers tobiomolecules can be eliminated, reducing any background emissionresulting from the conjugated polymer itself. The abovementionedbiomolecules include but are not limited to proteins, peptides, affinityligands, antibodies, antibody fragments, sugars, lipids, enzymes andnucleic acids (as hybridization probes and/or aptamers).

In general, in another aspect the invention includes the bioconjugationof polymers to affinity ligands (affinity ligands describing abiomolecule that has an affinity for another biomolecule). FIG. 2illustrates a class of materials in which a conjugated polymer (shown asa wavy line) is linked to a dye, biomolecule, or biomolecule/dye complex(labeled X). Linking to the conjugated polymer can be via a firstfunctionality linker A on the conjugated polymer that serves as abioconjugation site capable of covalently linking with a secondfunctionality linker A′ linked to a biomolecule and/or dye (see X). Thisarrangement can fix the distance between the conjugated polymer and X,thereby ensuring only specific interactions between polymer and X. It isenvisioned that a biomolecule component X in this embodiment can be anyof the various biomolecules disclosed herein, including but not limitedto an antibody, protein, affinity ligand, enzyme or nucleic acid.

Linker A can be anywhere on the conjugated polymer including terminalpositions of the polymer, internally on a repeating subunit, in betweenrepeating subunits or any combination thereof. Likewise, Linker A′ canbe linked anywhere on a biomolecule and/or dye. The linking chemistryfor A-A′ can include, but is not limited to, maleimide/thiol;thiol/thiol; pyridyldithiol/thiol; succinimidyl iodoacetate/thiol;N-succinimidylester (NHS ester), sulfodicholorphenol ester (SDP ester),or pentafluorophenyl-ester (PFP ester)/amine;bissuccinimidylester/amine; imidoesters/amines; hydrazine oramine/aldehyde, dialdehyde or benzaldehyde; isocyanate/hydroxyl oramine; carbohydrate-periodate/hydrazine or amine; diazirine/aryl azidechemistry; pyridyldithiol/aryl azide chemistry; alkyne/azide;carboxy-carbodiimide/amine; amine/Sulfo-SMCC (Sulfosuccinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate)/thiol; and amine/BMPH(N-[ß-Maleimidopropionic acid]hydrazide.TFA)/thiol.

It is envisioned that the X in this context can be, but is not limitedto, a dye, fluorescence protein, nanomaterial (e.g., Quantum Dot),chemiluminescence-generating molecule, a conjugate between dye andchemiluminescence-generating molecule, a conjugate between fluorescenceprotein and chemiluminescence-generating molecule, a conjugate betweennanomaterial (e.g., Quantum Dot) and chemiluminescence-generatingmolecule, streptavidin, avidin, enzyme, substrate for an enzyme,substrate analog for an enzyme, receptor, ligand for a receptor, ligandanalog for a receptor, DNA, RNA, modified nucleic acid, DNA aptamer, RNAaptamer, modified nucleic aptamer, peptide aptamer, antibody, antigen,phage, bacterium or conjugate of any two of the items described above.

In another aspect, the invention includes the use of conjugated polymersas direct labels. FIG. 3 shows examples of labeled conjugated polymers.In one embodiment, FIG. 3A, a polymer (shown as encircled hexagons) isshown conjugated to an antibody which can be, for example, a 1° or 2°antibody. The conjugate of the polymer and the antibody can be used as adirect reporter, for example, in an assay. In additional embodiments,the signal from the polymer is not modulated by other assay componentsrather it is dependent on its presence in the assay at the time ofdetection as a function of specific biomolecule recognition. Excitationof the polymer with light (not shown) can result in polymer emission,indicating the presence of the antibody (1° or 2°) in the assay or assaysolution. FIGS. 3B and 3C further exemplify the use of conjugatedpolymers as biomolecule labels capable of detecting specific targets andtarget associated biomolecules. FIG. 3B depicts a polymer avidin(streptavidin, neutraAvidin, etc.) conjugate capable of binding tobiotin modified molecules, biomolecules or substrates. FIG. 3C depicts anucleic acid (DNA, RNA, PNA, etc.) conjugate capable of hybridizing tocomplementary nucleic acid sequences. Linkage or conjugation offluorescent conjugated polymer to a molecule capable of recognizing atarget biomolecule or target associated molecule (such as thoseexemplified in FIG. 3) provides a direct means of detection. Inadditional embodiments, the signals generated from excitation of theconjugated polymer are not modulated by other assay components exceptthose which are directly conjugated to the polymer. In such embodimentsthe polymer complex is acting directly as a fluorescent label.

In another embodiment shown in FIG. 3D, the conjugated polymer islabeled with a dye, for example, a chromophore. In this case, theconjugated polymer can act as a donor and the dye can act as an acceptorin an energy transfer process. Here, the conjugated polymer can act as alight harvester, and excitation of the conjugated polymer is followed bythe channeling of the excitations to the dye via an energy transferprocess including, but not limited to, FRET. This results in amplifieddye emission (as compared to direct excitation of the dye). Thefluorescence of the donor conjugated polymer, in one embodiment, can bequenched (e.g., >90% quenching). This is exemplified in Example 38 andshown in FIG. 21, by way of example only. In some instances, theconjugated polymer in FIG. 3D (and similar drawings disclosed herein)can have multiple dye attachments which can be positioned internally orat the terminus of the polymer structure (single dye shown forillustrative purposes only).

In the case of direct linkage to a dye (FIG. 3D) or biomolecule/dyecomplex (as exemplified in FIG. 4), donor-acceptor distances can befixed, rather than dependent on the strength of interaction or binding,and energy transfer efficiency can be significantly increased. This hassignificant consequences in the context of improving dye signaling (orquenching) and reducing background fluorescence associated withdonor-acceptor cross-talk. Cross-talk in this case refers to the overlapbetween conjugated polymer (donor) and dye (acceptor) emission peaks.Conjugated polymers which bind non-specifically at distances too greatfor energy transfer can contribute to the background fluorescence (orcrosstalk). Shorter (fixed) distances between the donor and acceptor cannot only facilitate direct dye amplification, but also can greatlyquench the donor emission, as depicted in FIG. 21 by way of exampleonly. This results in less donor emission at the acceptor emissionwavelengths, which subsequently reduces or even eliminates the need forcross-talk correction.

In further embodiments the localization of the conjugated polymer and asignaling chromophore are brought together by recognition event, forexample by the binding of two affinity pairs or by co-recognition of thesame target molecule or target associated molecule (FIG. 5). Suchembodiments could be performed in solution based formats or in suchconfigurations where one or more of elements is bound to anotherbiomolecule (cell, tissue, protein, nucleic acid, etc.) or a substrate(bead, well plate, surface, tube, etc.).

In general, another aspect the invention includes a method of assayingfor a target biomolecule or target associated biomolecule. As shown inFIG. 5A, in one embodiment a conjugated polymer (shown as a wavy line)can be linked to a first bioconjugate (shown as a Y-shaped object), forexample, a 2° antibody that is specific for second a dye-labeledbioconjugate, for example, a 1° antibody. Here, the recognition eventbetween the 1° and 2° antibody will result in the reduction of distancebetween the donor conjugated polymer and acceptor dye. In a similarembodiment depicted in FIG. 5B, polymer and dye labeled antibodiesrecognize a common target. After either of these recognition events,excitation of the donor conjugated polymer with light (shown as hν) willresult in energy transfer, e.g., FRET, to the acceptor dye (shown ascurved arrow), and amplified dye emission (in comparison with directexcitation of the dye) will be observed. In use it is envisioned that anassay method could include providing a sample that is suspected ofcontaining a target biomolecule by the steps of providing a firstbioconjugate, for example, a 1° antibody conjugated to a signalingchromophore and capable of interacting with the target biomolecule. Thisis followed by providing a second bioconjugate, for example, a 2°antibody or 1° antibody, conjugated to a polymer, wherein the secondbioconjugate can bind to the first bioconjugate or target and whereinupon such binding excitation of the conjugated polymer is capable oftransferring energy to the signaling chromophore. Next, the methodincludes contacting the sample with the first bioconjugate in a solutionunder conditions in which the first bioconjugate can bind to the targetbiomolecule if present and contacting the solution with the secondbioconjugate. The method then includes applying a light source to thetarget biomolecule or tagged target biomolecule, wherein the lightsource can excite the conjugated polymer and subsequently detectingwhether light is emitted from the signaling chromophore.

In another aspect, the invention includes a method of assaying a sampleusing a conjugated polymer and a sensor biomolecule complex. As shown inFIGS. 5C and D, a polymer (shown as a wavy line) can be conjugated to afirst bioconjugate, for example, streptavidin (SA) which has a strongaffinity for biotin. In FIG. 5C, a sensor biomolecule (e.g., an antibodywhich can be a 1° or 2° antibody), is conjugated to both a dye and asecond bioconjugate (e.g., a biotin moiety). Similar embodiments aredepicted in FIG. 5D where the second bioconjugate (e.g., a biotinmoiety) and the signaling chromopohre are both conjugated to a nucleicacid. After a biorecognition event between the first and secondbioconjugates (e.g. between SA and biotin), the conjugated polymer anddye will be brought into close proximity, and excitation of the donorconjugated polymer will result in energy transfer to the acceptor dye.Dye emission will indicate the presence of the first bioconjugate (e.g.,the antibody or nucleic acid). In comparison with direct excitation ofthe dye, amplification of the dye signal intensity will be observed whenexcited indirectly through an energy transfer process, e.g., FRET.

A method of using the embodiment shown in FIGS. 5C and D can include thesteps of providing a sample that is suspected of containing a targetbiomolecule, providing a conjugated polymer comprising a covalentlylinked first bioconjugate (e.g., SA), providing a sensor biomoleculecomplex comprising a sensor biomolecule capable of interacting with thetarget molecule, a signaling chromophore, and covalently linked secondbioconjugate capable of binding with the first bioconjugate, whereinupon such binding excitation of the conjugated polymer is capable oftransferring energy to the signaling chromophore. The method can furtherinclude the steps of contacting the sample with the sensor biomoleculecomplex in a solution under conditions in which the sensor biomoleculecan bind to the target biomolecule if present, contacting the solutionwith the conjugated polymer, applying a light source to the sample thatcan excite the conjugated polymer, and detecting whether light isemitted from the signaling chromophore.

Further the conjugated polymer can contain additional linking sitesuitable for conjugation or attachment to more than one species. FIG. 6exemplifies the addition of a second linking site within the polymer.Such linkers A and B can be the same or different to allow fororthogonal conjugation of different species. The linkers can be anywhereon the polymer including terminal and internal positions. The linkingchemistry for A-A′ and B-B′ (and optionally C-C′, D-D′, etc.) caninclude, but is not limited to, maleimide/thiol; thiol/thiol;pyridyldithiol/thiol; succinimidyl iodoacetate/thiol;N-succinimidylester (NHS ester), sulfodicholorphenol ester (SDP ester),or pentafluorophenyl-ester (PFP ester)/amine;bissuccinimidylester/amine; imidoesters/amines; hydrazine oramine/aldehyde, dialdehyde or benzaldehyde; isocyanate/hydroxyl oramine; carbohydrate-periodate/hydrazine or amine; diazirine/aryl azidechemistry; pyridyldithiol/aryl azide chemistry; alkyne/azide;carboxy-carbodiimide/amine; amine/Sulfo-SMCC (Sulfosuccinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate)/thiol; and amine/BMPH(N-[ß-Maleimidopropionic acid]hydrazide.TFA)/thiol. A tri-functionallinker such as the commercially available Sulfo-SBEDSulfosuccinimidyl[2-6-(biotinamido)-2-(p-azidobenzamido)-hexanoamido]-ethyl-1,3′-dithiopropionatecan serve well in the three way linkage among X, Y, and conjugatedpolymer.

In the embodiment illustrated in FIG. 6, X or Y can be, but are notlimited to, a dye, fluorescence protein, nanomaterial (e.g., QuantumDot), chemiluminescence-generating molecule, a conjugate between dye andchemiluminescence-generating molecule, a conjugate between fluorescenceprotein and chemiluminescence-generating molecule, a conjugate betweennanomaterial (e.g., Quantum Dot) and chemiluminescence-generatingmolecule, streptavidin, avidin, enzyme, substrate for an enzyme,substrate analog for an enzyme, receptor, ligand for a receptor, ligandanalog for a receptor, DNA, RNA, modified nucleic acid, DNA aptamer, RNAaptamer, modified nucleic aptamer, peptide aptamer, antibody, antigen,phage, bacterium or conjugate of any two of the items described above.

In general, in another aspect the invention provides a conjugatedpolymer complex including a polymer, a sensor biomolecule and asignaling chromophore for identifying a target biomolecule. As shown inFIG. 6, in one embodiment a polymer (wavy line) can be bioconjugated toa dye X via linker functionalities A-A′ and a biomolecule Y via linkerfunctionalities B-B′. As depicted in FIG. 7, in one embodiment a polymercan be bioconjugated to both a dye and a biomolecule, for example abiorecognition molecule. Useful biomolecules can include but are notlimited to antibodies (FIG. 7A), avidin derivatives (FIG. 7B) affinityligands, nucleic acids (FIG. 7C), proteins, nanoparticles or substratesfor enzymes. The benefits of covalently linking a dye in proximity to apolymer have been described above. By affixing both an acceptor dye anda biorecognition molecule to a polymer, the benefits are two fold, byboth fixing donor-acceptor distances, such that an acceptor isguaranteed to be within the vicinity of a donor conjugated polymer (andvice versa), and also increasing the specificity of polymer binding toindicate a biorecognition event. These covalent complexes can be madevia the monomer, polymer and linking chemistries described herein.

In use, the embodiments shown in FIG. 6 can be a conjugated polymercomplex for identifying a target biomolecule wherein the complexincludes a conjugated polymer, a signaling chromophore covalently linkedto the conjugated polymer and a sensor biomolecule covalently linked tothe conjugated polymer. The signaling chromophore of the complex iscapable of receiving energy from the conjugated polymer upon excitationof the conjugated polymer and the sensor biomolecule is capable ofinteracting with the target biomolecule. It is envisioned that thebiomolecules can include but are not limited to an antibody, protein,affinity ligand, peptide, or nucleic acid.

In one embodiment shown in FIG. 7A, a polymer is conjugated to both abioconjugate, for example, an antibody (1° or 2°) and a dye. Covalentlinkage between the donor conjugated polymer and acceptor dye ensuresclose proximity. Excitation of the donor conjugated polymer results inenergy transfer, e.g., FRET, to the acceptor dye. Where the bioconjugateis an antibody, if the antibody binds to its target (e.g., antigen),this will be indicated by dye emission upon donor polymer excitation. Inan alternative embodiment, as shown in FIG. 7B, a polymer can beconjugated to both a SA and a dye. Again, covalent linkage between thedonor conjugated polymer and acceptor dye ensure close proximity, andexcitation of the donor conjugated polymer results in energy transfer tothe acceptor dye. The SA complex can be used to label or detect abiotin-labeled biomolecule such as a biotinylated antibody or nucleicacid. Polymer excitation followed by energy transfer to the dye labelwill result in greatly enhanced detection signals (i.e., greatersensitivity).

The example exemplified in FIG. 7A is a conjugated polymer labeled witha dye acceptor and further conjugated to an antibody. This Tandemconfiguration can be used in similar fashion as those described for thestructure in FIG. 3A but are useful in generating a secondary signal fordetection, often in multiplex formats. The conjugated polymer complexesin FIG. 7 can have multiple dye attachments which can be positionedinternally or at the terminus of the polymer structure (single dye shownfor illustrative purposes only).

In other embodiments as shown in FIGS. 3A and 7A, a sensor biomoleculefor example a 1° antibody (Y shape) is conjugated covalently linked tothe conjugated polymer (encircled hexagons) or conjugated polymer-dyetandem complex (hexagons with pendant encircled star). Upon conjugatedpolymer excitation, emission from the conjugated polymer (FIG. 3A) ordye (FIG. 7A) will indicate presence of the biocomplex and by extensionwith appropriate assay design that of the target recognized by thesensor molecule allowing use as a reporter, for example in an assay.FIGS. 29A and 29B represent comparable examples with covalent linkage ofthe conjugated polymer to a 2° antibody.

As an alternative embodiment, the conjugated polymer may be associatedindirectly with the sensor biomolecule or target associated biomolecule.FIGS. 8C and 8D illustrate a sequence specific oligonucleotide probe(wavy line) covalently conjugated to a biotin moiety (drop shape).

Here the conjugated polymer (encircled hexagons) or conjugatedpolymer-dye tandem complex (hexagons with pendant encircled star) iscovalently bound or conjugated to a biotin recognizing protein (forexample, avidin, streptavidin or similar with high specific affinity forthe ligand biotin). FIGS. 8A and 8B illustrate comparable examples witha biotinylated antibody interacting with a covalent conjugate of theconjugated polymer (FIG. 8A) and conjugated polymer-dye tandem complex(FIG. 8B) to the biotin recognizing protein. Indirect association of thetarget associated biomolecule with the conjugated polymer is not limitedto biotin mediated interactions. FIGS. 8E and F represent sequencespecific oligonucleotides (wavy line) which have been covalently labeledwith a digoxygenin moiety (7 pointed star). In turn the digoxygeninmoiety has been recognized by a primary antibody covalently linked tothe conjugated polymer (FIG. 8E) and the conjugated polymer-dye tandemcomplex (FIG. 8F). Although not shown pictorially, further embodimentsemploying indirect detection of digoxygenin using biotinylatedantibodies and biotin recognizing proteins covalently linked toconjugated polymers (or conjugated polymer-dye tandem complexes) orunlabelled primary antibodies recognizing digoxygenin and appropriatesecondary antibodies covalently linked to the conjugated polymer (orconjugated polymer-dye tandem complexes) are intended.

A number of further embodiments are also predicated on energy transfer(for example but not limited to FRET) between the conjugated polymer andan acceptor dye. Given the potential for multiplexing analysis, it isenvisioned that the conjugated polymer can be linked to a number of dyesor signaling chromophores, including, but not limited to, fluorescein,6-FAM, rhodamine, Texas Red, California Red, iFluor594,tetramethylrhodamine, a carboxyrhodamine, carboxyrhodamine 6G,carboxyrhodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow,coumarin, Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®, Cy7®, Cy-Chrome, DyLight350, DyLight 405, DyLight 488, DyLight 549, DyLight 594, DyLight 633,DyLight 649, DyLight 680, DyLight 750, DyLight 800, phycoerythrin, PerCP(peridinin chlorophyll-a Protein), PerCP-Cy5.5, JOE(6-carboxy-4′,5′-dichloro-2′,7′-dimelhoxyfluorescein), NED, ROX(5-(and-6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue,Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor® 350,Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor®546,Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 647,Alexa Fluor® 660, Alexa Fluor® 680, 7-amino-4-methylcoumarin-3-aceticacid, BODIPY® FL, BODIPY® FL-Br₂, BODIPY® 530/550, BODIPY® 558/568,BODIPY® 564/570, BODIPY® 576/589, BODIPY®581/591, BODIPY® 630/650,BODIPY® 650/665, BODPY® R6G, BODIPY® TMR, BODIPY® TR, conjugatesthereof, and combinations thereof. These embodiments includemodifications of the above examples where the acceptor dye serves as theassay reporter (as exemplified in FIGS. 3D, 4D, 7, 8B, 8D, 8E, 29B,wherein the encircled ten pointed star represents the dye).

In certain embodiments the conjugated polymer conjugates provided inFIGS. 2-10, 29 and 30 are intended for but not limited to use in flowcytometry, cell sorting, molecular diagnositics, fluorescence in situhybridization (FISH), immunohistochemistry (IHC), polymerase chainreaction, microscopy (fluorescent, confocal, 2 photon, etc.), blotting(e.g. northern, southern, western), cytomic bead arrays (Luminexformats, etc.), fluorescent immune assay (FIA or ELISA), nucleic acidsequencing and microarrays.

Embodiments are also envisaged where conjugated polymers are used toenhance the detection and quantification of nucleic acids using sequencespecific fluorescent probes combined with nucleic acid amplificationtechniques such as but not limited to polymerase chain reaction,transcription mediated amplification, rolling circle amplification,recombinase polymerase amplification, helicase dependent amplificationand Linear-After-The-Exponential polymerase chain reaction.

FIG. 32 represents modifications of the HybProbe detection technique. InFIG. 32A, the dye conventionally used as an energy transfer donor isreplaced by the conjugated polymer (hexagon chain) which is covalentlylinked to the donor probe (wavey helical structure represented as righthand helical duplex due to association with nucleic acid target depicteda longer helical wavy line). Upon sequence specific hybridization thedonor and acceptor (represented similarly to donor probe but on lefthand side of nucleic acid target) probes are spatially juxtaposed on thetarget nucleic acid strand of interest in sufficiently close proximityto allow energy transfer to take place between the fluors. Excitationenergy is transduced through the conjugated polymer and emitted as areadable signal by the dye (encircled ten pointed star) to allow nucleicacid quantification, detection and/or characterization. Presence ofincreased template allows increased numbers of probe co-hybridisationevents and thus correlates to increased specific signal from theacceptor dye. In combination with the melt curve technique commonlyemployed in HybProbe experiments it is envisaged that sequence specificinformation corresponding to sequence variations will be collectable inappropriately designed experiments. FIG. 32B represents a “signal off”modification of the HybProbe approach where the conjugated polymer isquenched by an acceptor probe consisting of a small moleculefluorescence quencher (for example but not limited to Black HoleQuenchers™, Iowa Black® or Dabsyl).

In another embodiment, conjugated polymer and conjugated polymer-dyetandem complexes similar to those described in FIGS. 4C and 4D are usedin the detection, quantification and/or characterization of nucleic acidtargets. Nucleic acid probe sequences labeled with a quencher molecule(black circle, for example but not limited to Black Hole Quenchers™,Iowa Black® or Dabsyl) are also conjugated to a conjugated polymer(FIGS. 4C and 31A) and a conjugated polymer-dye tandem complex (FIGS. 4Dand 31B). In FIGS. 4C and D the recognition of the target sequence leadsto a hybridization and separation of the quencher from the conjugatedpolymer or conjugated polymer-dye tandem complex and upon polymerexcitation produces an increase in fluorescent signal. In FIGS. 31A and31B the nucleic acid probe conjugate will hybridize to a complementarytarget sequence and by treatment with specific enzymes the probesequence is cleaved or hydrolyzed freeing the conjugated polymer orconjugated conjugated polymer-dye tandem complex from the quencher andupon polymer excitation produces an increase in fluorescent signal. Themost common example of the methods described in FIG. 31 is the use ofDNA polymerase enzymes which contain nuclease activity (e.g. TaqMan PCRassays).

FIG. 9 shows examples of conjugated polymer (hexagons) conjugated tosecondary antibodies (FIG. 9A) and primary antibodies (FIG. 9B)(antibodies shown as Y-shaped structures). In an assay, an unlabeled 1°antibody can bind to an antigen, for example, a target protein (shown asa black triangle). Addition of the 2° antibody, which is conjugated to apolymer, can bind specifically to the 1° antibody. After washing toremove unbound 2° antibody and upon application of light of suitableexcitation wavelength, observance of polymer emission is indicative ofspecific binding (FIG. 9A). In other assay embodiments, apolymer-labeled 1° antibody can directly bind a target protein, shown asa black triangle, and after washing to remove unbound 1° antibody andupon application of light of suitable excitation wavelength, observanceof polymer emission is indicative of specific binding (FIG. 9B).Optionally, whether conjugated to the 1° or 2° antibody, the polymer maybe further conjugated to a dye. In such a case, optical excitation ofthe conjugated polymer can result in energy transfer to the dye, andamplified dye emission, in comparison to direct dye excitation results.Observance of dye emission is indicative of specific binding.

FIG. 10 shows an example of a sandwich-type complex of one embodiment ofthe invention. In the assay shown in FIG. 10A the conjugated polymercomplex is composed of a polymer (shown as hexagons) that isbioconjugated a biomolecule, for example, streptavidin (X shape). Afteran unlabeled 1° antibody binds the target (e.g. protein), shown as ablack triangle, a biotin-labeled 2° antibody binds specifically to the1° antibody. In a separate step, addition of the conjugated polymercomplex will result in specific binding between the biotin andstreptavidin. Excitation of the conjugated polymer will result inpolymer emission, indicating the presence of the target protein.Additionally in another embodiment, a biotin-labeled 1° antibody may beused to probe the target protein directly (FIG. 10B). After this bindingevent takes place, addition of a streptavidin-polymer complex willresult in specific binding between the biotin and streptavidin, andexcitation of the conjugated polymer will result in polymer emission,indicating the presence of the target protein. Optionally, the polymermay be further conjugated to a dye. In such a case, optical excitationof the polymer will result in amplified dye emission, as compared todirect excitation of the dye. Signals arising from dye emission willindicate the presence of the target protein.

FIG. 30 depicts example embodiments around the use of conjugatedpolymers in Fluorescent Immuno Assay (FIA). In FIG. 30 panels A-Canalyte antigen is immobilised on a surface which can include but is notlimited to a microtitre plate well, bead particle, glass slide, plasticslide, lateral flow strip, laminar flow device, microfluidic device,virus, phage, tissue or cell surface. Analyte molecules are thendetected by use of labelled detection conjugates or sensor biomolecules.In FIG. 30A, a conjugated polymer covalently linked to a detectionantibody is utilized for detection. In FIG. 30B, a biotin bindingprotein (for example but not limited to avidin, streptavidin or otherhigh affinity biotin specific derivatives) covalently bound to theconjugated polymer and interacting with a biotinylated detectionantibody is utilized for detection. In FIG. 30C, a secondary antibodycovalently linked to the conjugated polymer and interacting with adetection antibody is utilized for detection. In FIG. 5B, a homogenous,solution based example is also embodied where two separate antibodieseach bind to the antigen of interest. One antibody is covalently linkedto the conjugated polymer, the other to a dye. When bound to theantigen, the respective fluorophores are brought into sufficient spatialproximity for energy transfer to occur. In assays predicated on thedesigns in FIG. 30 and FIG. 5B, the sample is interrogated with lightmatched to the excitation of the conjugated polymer and signal reportedat the emission wavelength of the dye. In the examples embodied in FIG.30 A-C the use of a polymer-dye tandem complex is further disclosed. Insuch cases, optical excitation of the polymer will result in amplifieddye emission, as compared to direct excitation of the dye. Signalsarising from dye emission will indicate the presence of the target.

In a further aspect, the invention provides for the multiplexing ofdonor energy transfer to multiple acceptors. By using a conjugatedpolymer as a donor in an energy transfer system, benefits also includethe ability to multiplex. A single donor can transfer energy to severaldyes; thus with a single excitation source, the intensity of multipledyes can be monitored. This is useful for applications including but notlimited to cell imaging (i.e. immunohistochemistry), flow cytometry andcell sorting, where the different types of cells can be monitored byprotein-antibody recognition events.

In one embodiment, two dye-labeled antibodies can be incubated with abiological material, for example, a cultured cell line, tissue sectionor blood sample. Antibodies are able to recognize cells with a targetprotein expressed on its surface and specifically bind only to thoseproteins. By labeling the two antibodies with different dyes, it ispossible to monitor for the expression of two different proteins ordifferent cell types simultaneously. Typically, this would require twoscans, excitations or images, once each with the correct excitationwavelength. As a final step prior to analysis, these two images or datasets would have to be overlaid or combined. By using antibodiesconjugated with both a dye and a conjugated polymer, one excitationwavelength can be used for the conjugated polymer to excite both dyes,and a single image or scan will include data sets from each of the twoantibodies. This can be done with any number of antibody combinationsprovided there is sufficient ability to resolve the resulting signals.

It is envisioned that the invention described herein can be used toincrease the sensitivity of any of a number of commercially availabletests including but not limited to the OraQuick Rapid HIV-1/2 AntibodyTest, manufactured by OraSure Technologies, Inc. (Bethlehem, Pa.), whichis a FDA-approved HIV diagnostic test for oral fluid samples. This testcan provide screening results with over 99 percent accuracy in as littleas 20 minutes.

Conjugated Polymers

Light harvesting conjugated polymer systems can efficiently transferenergy to nearby luminescent species. Mechanisms for energy transferinclude, for example, resonant energy transfer (Forster (orfluorescence) resonance energy transfer, FRET), quantum charge exchange(Dexter energy transfer) and the like. Typically, however, these energytransfer mechanisms are relatively short range, and close proximity ofthe light harvesting conjugated polymer system to the signalingchromophore is required for efficient energy transfer. Amplification ofthe emission can occur when the number of individual chromophores in thelight harvesting conjugated polymer system is large; emission from afluorophore can be more intense when the incident light (the “pumplight”) is at a wavelength which is absorbed by the light harvestingconjugated polymer system and transferred to the fluorophore than whenthe fluorophore is directly excited by the pump light.

The conjugated polymers used in the present invention can be chargeneutral, cationic or anionic. In some embodiments, the conjugatedpolymers are polycationic conjugated polymers. In other embodiments, theconjugated polymers are polyanionic conjugated polymers. In furtherembodiments, the conjugated polymers can include cationic, anionic,and/or neutral groups in various repeating subunits. In yet otherembodiments, the conjugated polymers are neutral conjugated polymers. Insome instances, conjugated polymers contain groups such as ethyleneglycol oligomers, ethylene glycol polymers, ω-ammonium alkoxy salts,and/or ω-sulfonate alkoxy salts that impart solubility in aqueoussolutions. In some instances the neutral conjugated polymers withnon-ionic side chains are soluble in greater than 10 mg/mL in water orphosphate buffered saline solutions and in certains cases the solubilityis greater than 50 mg/mL. In some embodiments the conjugated polymerscontain either a terminal linking site (e.g., capping unit), internallinking site or both.

In some embodiments, a conjugated polymer is one that comprises “lowbandgap repeat units” of a type and in an amount that contribute anabsorption to the polymer in the range of about 450 nm to about 1000 nm.The low bandgap repeat units may or may not exhibit such an absorptionprior to polymerization, but does introduce that absorption whenincorporated into the conjugated polymer. Such absorptioncharacteristics allow the polymer to be excited at wavelengths thatproduce less background fluorescence in a variety of settings, includingin analyzing biological samples and imaging and/or detecting molecules.Shifting the absorbance of the conjugated polymer to a lower energy andlonger wavelength thus allows for more sensitive and robust methods.Additionally, many commercially available instruments incorporateimaging components that operate at such wavelengths at least in part toavoid such issues. For example, thermal cyclers that perform real-timedetection during amplification reactions and microarray readers areavailable which operate in this region. Providing polymers that absorbin this region allows for the adaptation of detection methods to suchformats, and also allows entirely new methods to be performed.

Incorporation of repeat units that decrease the band gap can produceconjugated polymers with such characteristics. Exemplary optionallysubstituted species which result in polymers that absorb light at suchwavelengths include 2,1,3-benzothiadiazole, benzoxidazole,benzoselenadiazole, benzotellurodiazole, naphthoselenadiazole,4,7-di(thien-2-yl)-2,1,3-benzothiadiazole, squaraine dyes, quinoxalines,perylene, perylene diimides, diketopyrrolopyrrole, thienopyrazine lowbandgap commercial dyes, olefins, and cyano-substituted olefins andisomers thereof. Further details relating to the composition, structure,properties and synthesis of suitable conjugated polymers can be found inU.S. patent application Ser. No. 11/329,495, filed Jan. 10, 2006, nowpublished as US 2006-0183140 A1, which is incorporated herein byreference in the entirety.

In one aspect, provided herein are conjugated polymers of Formula (I):

wherein:

each R is independently a non-ionic side group capable of impartingsolubility in water in excess of 10 mg/mL;

MU is a polymer modifying unit or band gap modifying unit that is evenlyor randomly distributed along the polymer main chain and is optionallysubstituted with one or more optionally substituted substituentsselected from halogen, hydroxyl, C₁-C₁₂ alkyl, C₂-C₁₂ alkene, C₂-C₁₂alkyne, C₃-C₁₂ cycloalkyl, C₁-C₁₂ haloalkyl, C₁-C₁₂ alkoxy,C₂-C₁₈(hetero)aryloxy, C₂-C₁₈(hetero)arylamino,(CH₂)_(x′)(OCH₂CH₂)_(y′)OCH₃ where each x′ is independently an integerfrom 0-20, y′ is independently an integer from 0 to 50, or aC₂-C₁₈(hetero)aryl group;

each optional linker L₁ and L₂ are aryl or heteroaryl groups evenly orrandomly distributed along the polymer main chain and are substitutedwith one or more pendant chains terminated with a functional group forconjugation to another substrate, molecule or biomolecule selected fromamine, carbamate, carboxylic acid, maleimide, activated esters,N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azide,alkyne, aldehydes, thiols, and protected groups thereof;

G₁ and G₂ are each independently selected from hydrogen, halogen, amine,carbamate, carboxylic acid, maleimide, activated esters,N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azide,alkyne, aldehydes, thiol, optionally substituted aryl, optionallysubstituted heteroaryl, halogen substituted aryl, boronic acidsubstituted aryl, boronic ester substituted aryl, boronic esters,optionally substituted fluorine and aryl or heteroaryl substituted withone or more pendant chains terminated with a functional group, moleculeor biomolecule selected from amine, carbamate, carboxylic acid,carboxylate, maleimide, activated esters, N-hydroxysuccinimidyl,hydrazines, hydrazides, hydrazones, azide, alkyne, aldehydes, thiols,and protected groups thereof for conjugation to another substrate,molecule or biomolecule;

wherein the polymer comprises at least 1 functional group selected fromamine, carbamate, carboxylic acid, carboxylate, maleimide, activatedesters, N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones,azide, alkyne, aldehydes, and thiols within G₁, G₂, L₁ or L₂ thatallows, for functional conjugation to another molecule, substrate orbiomolecule;

each dashed bond, - - - - - - , is independently a single bond, triplebond or optionally substituted vinylene (—CR⁵═CR⁵—) wherein each R⁵ isindependently hydrogen, cyano, C₁-C₁₂ alkyl, C₂-C₁₂ alkene, C₂-C₁₂alkyne, C₃-C₁₂ cycloalkyl or a C₂-C₁₈(hetero)aryl group, wherein eachC₁-C₁₂ alkyl, C₂-C₁₂ alkene, C₂-C₁₂ alkyne, C₃-C₁₂ cycloalkyl or aC₂-C₁₈(hetero)aryl group is optionally substituted with one or morehalogen, hydroxyl, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl group,C₁-C₁₂ alkoxy, or C₁-C₁₂ haloalkyl; and

n is an integer from 1 to about 10,000; and

a, b, c and d define the mol % of each unit within the structure whicheach can be evenly or randomly repeated and where a is a mol % from 10to 100%, b is a mol % from 0 to 90%, and each c and d are mol % from 0to 25%.

Non-ionic side groups capable of imparting solubility in water as usedherein refer to side groups which are not charged and allow theresulting polymer to be soluble in water or aqueous solutions with novisible particulates. In some embodiments, each R is independently anon-ionic side group capable of imparting solubility in water in excessof 10 mg/mL, in excess of 15 mg/mL, in excess of 20 mg/mL, in excess of25 mg/mL, in excess of 30 mg/mL, in excess of 35 mg/mL, in excess of 40mg/mL, in excess of 45 mg/mL, in excess of 50 mg/mL, in excess of 60mg/mL, in excess of 70 mg/mL, in excess of 80 mg/mL, in excess of 90mg/mL or in excess of 100 mg/mL.

In some embodiments, conjugated polymers described herein comprises aminimum number average molecular weight of greater than 5,000 g/mol,greater than 10,000 g/mol, greater than 15,000 g/mol, greater than20,000 g/mol, greater than 25,000 g/mol, greater than 30,000 g/mol,greater than 40,000 g/mol, greater than 50,000 g/mol, greater than60,000 g/mol, greater than 70,000 g/mol, greater than 80,000 g/mol,greater than 90,000 g/mol, or greater than 100,000 g/mol.

In some embodiments, each R is independently (CH₂)_(x)(OCH₂CH₂)_(y)OCH₃where each x is independently an integer from 0-20, each y isindependently an integer from 0 to 50, or a benzyl optionallysubstituted with one or more halogen, hydroxyl, C₁-C₁₂ alkoxy, or(OCH₂CH₂)_(z)OCH₃ where each z is independently an integer from 0 to 50.In some instances, each R is (CH₂)₃(OCH₂CH₂)₁₁OCH₃.

In other embodiments, each R is independently a benzyl substituted withat least one (OCH₂CH₂)_(z)OCH₃ group where each z is independently aninteger from 0 to 50. In some instances, each R is a benzyl substitutedwith at least one (OCH₂CH₂)₁₀OCH₃ group. In other instances, each R is abenzyl substituted with at least two (OCH₂CH₂)₁₀OCH₃ groups. In furtherinstances, each R is a benzyl substituted with at least three(OCH₂CH₂)₁₀OCH₃ groups.

In further embodiments, each R is independently

where k and l are independent integers from 0 to 25;

*=site for covalent attachment.

In yet further embodiments, each R is independently is a dendrimer ofPAMAM, PEA, PEHAM, PPI, tri-branched benzoate, or glycerol with ageneration of 1 to 4 and optionally terminal substitutions, saidoptionally terminal substitutions are ( - - - - - )(CH₂CH₂O)_(j)CH₃ or( - - - - - )(OCH₂CH₂)_(j)CH₃ and j is an integer from 0 to 25 and thedotted lines ( - - - - - ) are each independently selected from any oneor a combination of, C₁-C₁₂ alkyl, C₁-C₁₂ alkoxy, C₂-C₁₂ alkene, amido,amino, aryl, (CH₂)_(r)(OCH₂CH₂)_(s)(CH₂)_(r) where each r isindependently an integer from 0-20, s is independently an integer from 0to 50, carbamate, carboxylate, C₃-C₁₂ cycloalkyl, imido, phenoxy, orC₄-C₁₈(hetero)aryl groups.

In alternative embodiments, each R is independently,

Where k and l are independent integers from 0 to 25 and the dotted lines( - - - - - ) are each independently selected from any one or acombination of, C₁-C₁₂ alkyl, C₁-C₁₂ alkoxy, C₂-C₁₂ alkene, amido,amino, aryl, (CH₂)_(r)(OCH₂CH₂)_(s)(CH₂)_(r) where each r isindependently an integer from 0-20, s is independently an integer from 0to 50, carbamate, carboxylate, C₃-C₁₂ cycloalkyl, imido, phenoxy, orC₄-C₁₈(hetero)aryl groups; *=site for covalent attachment.

In alternative embodiments, each R is independently,

Where k and l are independent integers from 0 to 25 and the dotted lines( - - - - - ) are each independently selected from any one or acombination of, C₁-C₁₂ alkyl, C₁-C₁₂ alkoxy, C₂-C₁₂ alkene, amido,amino, aryl, (CH₂)_(r)(OCH₂CH₂)_(s)(CH₂)_(r) where each r isindependently an integer from 0-20, s is independently an integer from 0to 50, carbamate, carboxylate, C₃-C₁₂ cycloalkyl, imido, phenoxy, orC₄-C₁₈(hetero)aryl groups; *=site for covalent attachment.

In some embodiments, conjugated polymers described herein contain nooptional linkers, L₁ and/or L₂. In other embodiments, conjugatedpolymers contain at least about 0.01 mol %, at least about 0.02 mol %,at least about 0.05 mol %, at least about 0.1 mol %, at least about 0.2mol %, at least about 0.5 mol %, at least about 1 mol %, at least about2 mol %, at least about 5 mol %, at least about 10 mol %, at least about20 mol %, or about 25 mol % of optional linkers, L₁ and/or L₂. In someembodiments, conjugated polymers contain up to 50 mol % total ofoptional linkers, L₁ and L₂, and may contain about 40 mol % or less,about 30 mol % or less, about 25 mol % or less, about 20 mol % or less,about 15 mol % or less, about 10 mol % or less, or about 5 mol % orless. Linkers can be evenly or randomly distributed along the polymermain chain.

In some embodiments, optional linkers L₁ or L₂ have the structure:

*=site for covalent attachment to unsaturated backbone

wherein R³ is independently hydrogen, halogen, hydroxyl, C₁-C₁₂ alkyl,C₂-C₁₂ alkene, C₂-C₁₂ alkyne, C₃-C₁₂ cycloalkyl or a C₂-C₁₈(hetero)arylgroup, wherein each C₁-C₁₂ alkyl, C₂-C₁₂ alkene, C₂-C₁₂ alkyne, C₃-C₁₂cycloalkyl or a C₂-C₁₈(hetero)aryl group is optionally substituted withone or more halogen, hydroxyl, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂alkynyl group, C₁-C₁₂ alkoxy, or C₁-C₁₂ haloalkyl; and q is an integerfrom 0 to 4.

In some embodiments, optional linkers L₁ or L₂ have the structurerepresented by:

*=site for covalent attachment to unsaturated backbonewherein A is a site for conjugation, chain extension or crosslinking andis —[O—CH₂—CH₂]t-W, or (C₁-C₁₂)alkoxy-X;

W is —OH or —COOH;

X is —NH₂, —NHCOOH, —NHCOOC(CH₃)₃,—NHCO(C₃-C₁₂)cycloalkyl(C1-C4)alkyl-N-maleimide; or—NHCO[CH₂—CH₂—O]_(u)NH₂;

t is an integer from 1 to 20; and

u is an integer from 1 to 8.

In other embodiments, optional linkers L₁ or L₂ are selected from thegroup consisting of a-h having the structure:

-   -   *=site for covalent attachment to unsaturated backbone

wherein R′ is independently H, halogen, C₁-C₁₂ alkyl, (C₁-C₁₂ alkyl)NH₂,C₂-C₁₂ alkene, C₂-C₁₂ alkyne, C₃-C₁₂ cycloalkyl, C₁-C₁₂ haloalkyl,C₂-C₁₈(hetero)aryl, C₂-C₁₈(hetero)arylamino, —[CH₂—CH₂]_(r′)—Z¹, or(C₁-C₁₂)alkoxy-X¹; and wherein Z¹ is —OH or —COOH; X¹ is —NH₂, —NHCOOH,—NHCOOC(CH₃)₃, —NHCO(C3-C12)cycloalkyl(C1-C4)alkyl-N-maleimide; or—NHCO[CH₂—CH₂—O]_(s′)(CH₂)_(s′)NH₂; r′ is an integer from 1 to 20; andeach s′ is independently an integer from 1 to 20,(CH₂)₃(OCH₂CH₂)_(x″)OCH₃ where x″ is independently an integer from 0 to50, or a benzyl optionally substituted with one or more halogen,hydroxyl, C₁-C₁₂ alkoxy, or (OCH₂CH₂)_(y″)OCH₃ where each y″ isindependently an integer from 0 to 50 and R′ is different from R;

wherein R¹⁵ is selected from the group consisting of l-t having thestructure:

and k is 2, 4, 8, 12 or 24; *=site for covalent attachment to backbone.

In certain embodiments, optional linkers L₁ or L₂ are

In some embodiments, G₁ and G₂ are optionally substituted aryl whereinthe optional substituent is selected from halogen, amine, carbamate,carboxylic acid, maleimide, activated esters, N-hydroxysuccinimidyl,hydrazines, hydrazides, hydrazones, azide, alkyne, aldehydes, boronicacid, boronate radical, boronic esters and optionally substitutedfluorene.

In other embodiments, G₁ and G₂ are the same. In further embodiments, G¹and G² are different. G¹ and G² can be activated units that allowfurther conjugation, crosslinking, or polymer chain extension, or theymay be nonactivated termination units.

In some embodiments, G₁ and G₂ are independently selected fromstructures represented by:

-   -   *=site for covalent attachment to backbone    -   wherein R¹¹ is any one of or a combination of a bond, C₁-C₂₀        alkyl, C₁-C₂₀ alkoxy, C₂-C₂₀ alkene, C₂-C₂₀ alkyne, C₃-C₂₀        cycloalkyl, C₁-C₂₀ haloalkyl, (CH₂)_(x)(OCH₂CH₂)_(p)(CH₂)_(x)        where each x is independently an integer from 0-20, p is        independently an integer from 0 to 50, aryl, C₂-C₁₈(hetero)aryl,        phenoxy, amido, amino, carbamate, carboxylate, carbonates,        sulfide, disulfide, or imido groups terminated with a functional        group selected from amine, carbamate, carboxylate, carboxylic        acid, maleimide, activated esters, N-hydroxysuccinimidyl,        hydrazines, hydrazides, hydrazones, azide, alkyne, aldehydes,        thiols, and protected groups thereof for conjugation to another        substrate, molecule or biomolecule.

In other embodiments, G₁ and G₂ are independently selected from thegroup consisting of 1-18 having the structure:

-   -   *=site for covalent attachment to backbone        wherein R¹⁵ is selected from the group consisting of l-t having        the structure:

-   -   and k is 2, 4, 8, 12 or 24.

In further embodiments, G₁ and G₂ is

In some embodiments, optional linkers, L₁ and/or L₂, G₁, and/or G₂ canbe further conjugated to an organic dye, a biomolecule or a substrate.Covalent linkage can be introduced by any known method and can include,but is not limited to, chemistry involving maleimide/thiol; thiol/thiol;pyridyldithiol/thiol; succinimidyl iodoacetate/thiol;N-succinimidylester (NHS ester), sulfodicholorphenol ester (SDP ester),or pentafluorophenyl-ester (PFP ester)/amine;bissuccinimidylester/amine; imidoesters/amines; hydrazine oramine/aldehyde, dialdehyde or benzaldehyde; isocyanate/hydroxyl oramine; carbohydrate-periodate/hydrazine or amine; diazirine/aryl azidechemistry; pyridyldithiol/aryl azide chemistry; alkyne/azide;carboxy-carbodiimide/amine; amine/Sulfo-SMCC (Sulfosuccinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate)/thiol; and amine/BMPH(N-[ß-Maleimidopropionic acid]hydrazide.TFA)/thiol.

In some embodiments, MU is selected from the group consisting of a′-k′having the structure:

=site for covalent attachment to unsaturated backbonewherein R is a non-ionic side group capable of imparting solubility inwater in excess of 10 mg/mL. Non-ionic side groups include thosepreviously described for polymers of Formula (I).

As used herein, in some embodiments, a pendant chain is any one of or acombination of a bond, C₁-C₂₀ alkyl, C₁-C₂₀ alkoxy, C₂-C₂₀ alkene,C₂-C₂₀ alkyne, C₃-C₂₀ cycloalkyl, C₁-C₂₀ haloalkyl,(CH₂)_(x)(OCH₂CH₂)_(p)(CH₂)_(x) where each x is independently an integerfrom 0-20, p is independently an integer from 0 to 50, aryl,C₂-C₁₈(hetero)aryl, phenoxy, amido, amino, carbamate, carboxylate,carbonates, sulfide, disulfide, or imido groups which connects a polymerwith a functional group for conjugation to another substrate, molecule,or biomolecule.

In some embodiments, conjugated polymers of Formula (I) have thestructure of Formula (Ia):

wherein R, L₁, L₂, G₁, G₂, MU, a, b, c, d and n are described previouslyfor formula (I).

In a further aspect, conjugated polymers of Formula I have the structureof Formula (Ib):

wherein at least one of G₁ or G₂ comprises a functional conjugationcite.

In a further aspect, conjugated polymers of Formula I have the structureof Formula (Ic):

wherein L₁ comprises a functional conjugation cite.

In a further aspect, conjugated polymers of Formula I have the structureof Formula (Id):

wherein at least one of G₁ or G₂ comprises a functional conjugationcite.

In a further aspect, conjugated polymers of Formula I have the structureof Formula (II):

wherein L₁, G₁, G₂, a, c, n and dashed bonds are described previouslyfor formula (I).

In some embodiments, conjugated polymers of Formula (II) have thestructure of Formula (IIa):

wherein L₁, G₁, G₂, a, c, and n are described previously for formula(I).

In a further aspect, conjugated polymers of Formula I have the structureof Formula (III):

wherein L₁, G₁, G₂, a, c, n and dashed bonds are described previouslyfor formula (I).

In some embodiments, conjugated polymers of Formula (III) have thestructure of Formula (IIIa):

wherein L₁, G₁, G₂, a, c, and n are described previously for formula(I).

In a further aspect, conjugated polymers of Formula I have the structureof Formula (IV):

wherein each R⁵ is independently hydrogen, cyano, C₁-C₁₂ alkyl, C₂-C₁₂alkene, C₂-C₁₂ alkyne, C₃-C₁₂ cycloalkyl or a C₂-C₁₈(hetero)aryl group,wherein each C₁-C₁₂ alkyl, C₂-C₁₂ alkene, C₂-C₁₂ alkyne, C₃-C₁₂cycloalkyl or a C₂-C₁₈(hetero)aryl group is optionally substituted withone or more halogen, hydroxyl, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂alkynyl group, C₁-C₁₂ alkoxy, or C₁-C₁₂ haloalkyl; and

L₁, G₁, G₂, a, c, n and dashed bonds are described previously forformula (I).

In a further aspect, conjugated polymers of Formula I have the structureof Formula (V):

wherein each R⁵ is independently hydrogen, cyano, C₁-C₁₂ alkyl, C₂-C₁₂alkene, C₂-C₁₂ alkyne, C₃-C₁₂ cycloalkyl or a C₂-C₁₈(hetero)aryl group,wherein each C₁-C₁₂ alkyl, C₂-C₁₂ alkene, C₂-C₁₂ alkyne, C₃-C₁₂cycloalkyl or a C₂-C₁₈(hetero)aryl group is optionally substituted withone or more halogen, hydroxyl, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂alkynyl group, C₁-C₁₂ alkoxy, or C₁-C₁₂ haloalkyl; and

L₁, G₁, G₂, a, c, n and dashed bonds are described previously forformula (I).

Also provided herein are polymers having the structure of the followingformula:

wherein: G¹ and G² are each independently selected from hydrogen,halogen, amine, carbamate, carboxylic acid, maleimide, activated esters,N-hydroxysuccinimidyl, hydrazines, hydrazides, hydrazones, azide,alkyne, aldehydes, optionally substituted aryl, halogen substitutedaryl, boronic acid substituted aryl, boronic ester substituted aryl,boronic esters and optionally substituted fluorene; L is a bond or anaryl or heteroaryl group that is evenly or randomly distributed alongthe polymer main chain and is optionally substituted with one or moreoptionally substituted substituents selected from halogen, hydroxyl,C₁-C₁₂ alkyl, C₂-C₁₂ alkene, C₂-C₁₂ alkyne, C₃-C₁₂ cycloalkyl, C₁-C₁₂haloalkyl, C₁-C₁₂ alkoxy, C₂-C₁₈(hetero)aryloxy,C₂-C₁₈(hetero)arylamino, (CH₂)_(x)(OCH₂CH₂)_(p)OCH₃ where each x isindependently an integer from 0-20, p is independently an integer from 0to 50, or a C₂-C₁₈(hetero)aryl group; L¹, L^(1′), L² and L^(2′)are eachindependently a covalent bond, a C1-C12 alkylene, a C3-C12cycloalkylene, a C₂-C₁₂ alkenylene, a C₂-C₁₂ alkynylene, a(C₆-C₁₈)aryl(C₁-C₁₂)alkylene, a (C₆-C₁₈)aryl(C₂-C₁₂)alkenylene, a(C₆-C₁₈)aryl(C₁-C₁₂)alkynylene, a C₆-C₁₈ arylene group, —Y₁—[O—Y₂]_(p)—,—O—Y₁—[O—Y₂]_(p)— wherein each C₁-C₁₂ alkylene, C₃-C₁₂ cycloalkylene,(C₆-C₁₈)aryl(C₁-C₁₂)alkylene, or C₆-C₁₈ arylene group is optionallysubstituted with one or more halogen, hydroxyl, C₁-C₁₂ alkyl, C₂-C₁₂alkenyl, C₂-C₁₂ alkynyl group, C₁-C₁₂ alkoxy, C₁-C₁₂ haloalkyl,—Y₁—[O—Y₂]_(p)— or —O—Y₁—[O—Y₂]_(p)—; q is 0 or an integer from 1 to 8;p is 0 or an integer from 1 to 24; Y₁ and Y₂ are each independently acovalent bond, or a C₁₋₁₂ alkylene group, a C₃-C₁₂ cycloalkylene, aC₂-C₁₈(hetero)arylene, a (C₆-C₁₈)aryl(C₁-C₁₂)alkylene, wherein eachC₁₋₁₂ alkylene group, a C₃-C₁₂ cycloalkylene, a C₂-C₁₈(hetero)arylene, a(C₆-C₁₈)aryl(C₁-C₁₂)alkylene is optionally substituted with one or morehalogen, hydroxyl, C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl group,C₁-C₁₂ alkoxy, or C₁-C₁₂ haloalkyl; E¹ and E_(1′) are eachindependently, hydrogen, C1-C6 alkyl, —OH, —COOH, —SH, —SR, —SHR⁺, SR₂⁺, —SO₃ ⁻, —PO₄ ⁻, Br, —NH₂, —NHR, —NR₂, —NH₃ ⁺, —NH₂R⁺, —NHR₂ ⁺ or —NR₃⁺, wherein and each R is independently a C₁-C₆ alkyl and —SHR⁺, SR₂ ⁺,—SO₃ ⁻, —PO₄ ⁻, —NH₃ ⁺, —NH₂R⁺, —NHR₂ ⁺ or —NR₃ ⁺ each optionally has anassociated counterion; and n is an integer from 1 to about 1,000.

Also provided herein are polymers having the structure of the followingformula:

wherein each R is independently O(CH_(x)), or (CH₂)₃(OCH₂CH₂)_(p)OCH₃where each x is independently an integer from 0-20, each p isindependently an integer from 0 to 50, or a benzyl optionallysubstituted with one or more halogen, hydroxyl, C₁-C₁₂ alkoxy, or(OCH₂CH₂)mOCH₃ where each m is independently an integer from 0 to 50; G1is selected from hydrogen, halogen, amine, carbamate, carboxylic acid,maleimide, activated esters, N-hydroxysuccinimidyl, hydrazines,hydrazides, hydrazones, azide, alkyne, aldehydes, optionally substitutedaryl, halogen substituted aryl, boronic acid substituted aryl, boronicester substituted aryl, boronic esters and optionally substitutedfluorene; and n is an integer from 1 to about 10,000.

Additional embodiments of conjugated polymers are described in thefollowing Examples.

Preparation of Conjugated Polymers

The synthesis of conjugated polymers described herein may beaccomplished using means described in the chemical literature, using themethods described herein, or a combination thereof.

Conjugated polymers described herein may be synthesized using standardsynthetic techniques known to those of skill in the art or using methodsknown in the art in combination with methods described herein. Inadditions, solvents, temperatures and other reaction conditionspresented herein may vary according to the practice and knowledge ofthose of skill in the art.

The starting material used for the synthesis of the conjugated polymersof Formula (1) and polymers having the structures described in the priorsection as described herein can be obtained from commercial sources,such as Aldrich Chemical Co. (Milwaukee, Wis.), Sigma Chemical Co. (St.Louis, Mo.), or the starting materials can be synthesized. The polymersdescribed herein, and other related polymers having differentsubstituents can be synthesized using techniques and materials known tothose of skill in the art, such as described, for example, in March,ADVANCED ORGANIC CHEMISTRY 4^(th) Ed., (Wiley 1992); Carey and Sundberg,ADVANCED ORGANIC CHEMISTRY 4^(th) Ed., Vols. A and B (Plenum 2000,2001), and Green and Wuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS 3′Ed., (Wiley 1999) (all of which are incorporated by reference in theirentirety). General methods for the preparation of polymers as disclosedherein may be derived from known reactions in the field, and thereactions may be modified by the use of appropriate reagents andconditions, as would be recognized by the skilled person, for theintroduction of the various moieties found in the formulae as providedherein. As a guide the following synthetic methods may be utilized.

Generally, polymerization of fluorene polymeric structures may beaccomplished using polymerization techniques known to those of skill inthe art or using methods known in the art in combination with methodsdescribed herein. For example, polymerization can be achieved via Suzukicoupling with a commercially available fluorene-dihalide monomer, e.g.,2,7-dibromofluorene, and its diboronic acid or ester derivative:

Structures A-1 and A-2 are catalyzed by a metal catalyst to formexemplary polymer A-3 with termination points, labeled Y. Each Y isindependently —H, —Br, —B(OH)₂, or boronic ester, e.g.,4,4,5,5,-tetramethyl-1,3,2-dioxaborolanyl.

Synthesis of diboronic ester derivatives from a fluorene-dihalidemonomer can also be accomplished via Suzuki coupling withbis(pinacolato)diboron:

Substituents such as ethylene glycol oligomers or ethylene glycolpolymers may be attached to monomers prior to polymerization or to thepolymer itself after polymerization. An exemplary scheme of synthesizingsubstituted fluorene monomers with mPEGylated groups is as follows:

2,7-dibromofluorene (B-1) and 3-bromopropanol in the presence of astrong base such as sodium hydroxide, potassium hydroxide, or the likeand a phase transfer catalyst, e.g. tetrabutylammonium bromide, isheated and reacted to completion to form2,7-dibromo-9,9-di(3′-hydroxypropanyl)fluorene (B-2). —OH groups of B-2are tosylated with tosyl chloride in the presence of pyridine andallowed to react to completion to form2,7-dibromo-9,9-di(3′-methylbenzenesulfonatopropanyl)fluorene (B-3). B-3is then reacted with a mPEG(x) alcohol in the presence of potassiumtert-butoxide to form B-4 with attached mPEG chains. mPEG alcohols canhave 1-50 mPEG chains. Typical sizes include but are not limited tomPEG5, mPEG8, mPEG11, mPEG24. In an alternative scheme, mPEG alcoholscan be tosylated first via tosyl chloride and then reacted to B-2 toform B-4.

Substituted monomers, such as exemplary structure B-4, can be furtherderivatized to diboronic esters in the schemes disclosed herein andsubsequently be used for polymerization such as via Suzuki coupling.Polymeric fluorenes may also be obtained through the use of otherreaction schemes involving organometallic catalysis. For example, theYamamoto reaction uses a nickel(0)-based catalyst for the homo-couplingof aryl halide monomers like exemplary structure B-4. Additionally,conjugated polymers can be synthesized using Stille, Heck, andSonogashira coupling reactions. See, e.g., Yamamoto et al.,Macromolecules 25: 1214-1223, 1992; Kreyenschmidt et al., Macromolecules28: 4577-4582, 1995; and Pei et al., J. Am. Chem. Soc. 118: 7416-7417,1996 regarding Yamamoto reaction schemes. See, also, Leclerc, Polym.Sci. Part A: Polym. Chem. 39: 2867-2873, 2001 for Stille reactionschemes; Mikroyannidis et al., J. Polym. Sci. Part A: Polym. Chem. 45:4661-4670, 2007 for Heck reaction schemes; and Sonogashira et al.,Tetrahedron Lett. 16: 4467-4470, 1975 and Lee et al., Org. Lett. 3:2005-2007, 2001 for Sonogashira reaction schemes.

Linkers and capping units can be conjugated to a fluorene polymerbackbone via similar mechanisms as described previously. For example,bromo- and boronic esters of capping units can be used to append one orboth ends of a polymer. Utilizing both bromo- and boronic esters ofcapping units will append both ends of polymer. Utilizing only one form,either a bromo- or boronic ester of a capping unit, will append onlythose ends terminated with its respective complement and for symmetricA-A+B-B polymerizations can be used to statistically modify only one endof a polymer. For asymmetric polymers this approach is used tochemically ensure the polymers are only modified at a single chainterminus. FIG. 11 depicts appending an exemplary fluorene polymer with Yends with one or more phenyl groups with bromobenzene, phenyl boronicacid or both using Suzuki coupling.

Capping units can also be appended asymmetrically by first reacting abromo-capping unit with a polymer with Y ends and subsequently reactingthe polymer with a boronic ester capping unit. Exemplary bromo- andboronic ester capping units include but are not limited to the followingstructures:

Further capping units can be found in structures 1-31 described hereinor in the following Examples and methods for their attachment.

The incorporation of optional linkers into conjugated polymer backbonesfurther described in U.S. application Ser. No. 11/868,870, filed Oct. 8,2007 and published as U.S. Application No. 2008/0293164, whichapplication is herein incorporated by reference in its entirety.

A desired optional linker incorporation can be achieved by varying themolar ratio of optional linker to bi-functional monomer. For example, anoptional linker can be incorporated by substituting a percentage of oneof the bi-functional monomers with a similar bi-functional optionallinker which comprises the conjugation site of interest. The number andtype of linking site included in the polymer is controlled by the feedratio of the monomers to optional linker in the polymerization reaction.By varying the feed ratio, conjugated polymers can contain at leastabout 0.01 mol % of linker, L, and may contain at least about 0.02 mol%, at least about 0.05 mol %, at least about 0.1 mol %, at least about0.2 mol %, at least about 0.5 mol %, at least about 1 mol %, at leastabout 2 mol %, at least about 5 mol %, at least about 10 mol %, at leastabout 20 mol %, or at least about 30 mol %. The conjugated polymers maycontain up to 100 mol % of linker, L, and may contain about 99 mol % orless, about 90 mol % or less, about 80 mol % or less, about 70 mol % orless, about 60 mol % or less, about 50 mol % or less, or about 40 mol %or less. Linkers can be evenly or randomly distributed along the polymermain chain. In further embodiments, an optional linker can further allowcovalent attachment of the resulting polymer to biomolecules, secondaryreporters or other assay components.

In alternative embodiments, the methods described herein to incorporateoptional linkers may be used in combination with methods of introducingcapping units with linking sites to produce polymers with both internaland terminal linking sites for conjugation. A non-limiting applicationof a polymer with both optional linkers and terminal capping units withlinking sites for conjugation are polymer-dye-biomolecule tandemconjugates where the polymer is used as an energy transfer donor, suchas in FRET, to a secondary dye acceptor thus shifting the emissionwavelength to that of the corresponding dye.

The person skilled in the art may further appreciate various synthesesand polymerization methods and embodiments of the present disclosureupon review of the following illustrative and non-limiting Examples.

Antigen-Antibody Interactions

The interactions between antigens and antibodies are the same as forother noncovalent protein-protein interactions. In general, four typesof binding interactions exist between antigens and antibodies: (i)hydrogen bonds, (ii) dispersion forces, (iii) electrostatic forcesbetween Lewis acids and Lewis bases, and (iv) hydrophobic interactions.Certain physical forces contribute to antigen-antibody binding, forexample, the fit or complimentary of epitope shapes with differentantibody binding sites. Moreover, other materials and antigens maycross-react with an antibody, thereby competing for available freeantibody.

Measurement of the affinity constant and specificity of binding betweenantigen and antibody is a pivotal element in determining the efficacy ofan immunoassay, not only for assessing the best antigen and antibodypreparations to use but also for maintaining quality control once thebasic immunoassay design is in place.

Antibodies

Antibody molecules belong to a family of plasma proteins calledimmunoglobulins, whose basic building block, the immunoglobulin fold ordomain, is used in various forms in many molecules of the immune systemand other biological recognition systems. A typical immunoglobulin hasfour polypeptide chains, containing an antigen binding region known as avariable region and a non-varying region known as the constant region.

Native antibodies and immunoglobulins are usually heterotetramericglycoproteins of about 150,000 Daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (VH) followed by a number of constant domains. Eachlight chain has a variable domain at one end (VL) and a constant domainat its other end. The constant domain of the light chain is aligned withthe first constant domain of the heavy chain, and the light chainvariable domain is aligned with the variable domain of the heavy chain.

Depending on the amino acid sequences of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are at least five (5) major classes of immunoglobulins: IgA, IgD,IgE, IgG and IgM, and several of these may be further divided intosubclasses (isotypes), e.g. IgG-1, IgG-2, IgG-3 and IgG-4; IgA-1 andIgA-2. The subunit structures and three-dimensional configurations ofdifferent classes of immunoglobulins are well known. Further detailsregarding antibody structure, function, use and preparation arediscussed in U.S. Pat. No. 6,998,241, issued Feb. 14, 2006, the entirecontents of which are incorporated herein by reference.

Sandwich Assays

Antibody or multiple antibody sandwich assays are well known to thoseskilled in the art including a disclosed in U.S. Pat. No. 4,486,530,issued Dec. 4, 1984, and references noted therein. The structuresdescribed in FIGS. 6, 7, 8, 9, 10 and 14 can be used directly asdescribed or in various sandwich configurations including thosedescribed in Example 37. A sandwich configuration or a sandwich assayrefers to the use of successive recognition events to build up layers ofvarious biomolecules and reporting elements to signal the presence of aparticular biomolecule, for example a target biomolecule or atarget-associated biomolecule. A standard example of this would be thesuccessive use of antibodies. In these assays, a primary antibody bindsthe target, the secondary antibody binds the primary, a third antibodycan bind the secondary and so on. With each successive layer additionalreporting groups can be added. Another strategy is using a repetitiveaddition of alternating layers of two (or more) mutually-recognizablecomponents, or more than two components in a chain-recognitionrelationship, which comprise one or both of the components in a form ofmultimeric structure. In such a setup, one or more of the functionalgroup(s) in each of the multimeric structure can be labeled withreporting group(s) and the unoccupied functional group(s) can serve asthe recognition site for the other component(s), and this system willsubsequently provide a platform for signal amplification. A typicalexample of this approach is the use of streptavidin-reporter conjugateand biotinylated anti-streptavidin antibody. In such assays, abiotinylated sensor molecule (nucleic acid or antibody) can be used tobind a target biomolecule, which is subsequently recognized by adetection system containing a streptavidin-reporter conjugate andbiotinylated anti-streptavidin antibody. The sandwich structure in thiscase can be built up by successive rounds of biotinylated antibodies andlabeled streptavidin complexes interaction to achieve the signalamplification. With an additional conjugation of a conjugated polymer toeither the biotinylated antibody or the streptavidin-reporter complex,it is possible to further increase the signal output. In essence, theintegration of a conjugated polymer in this type of signal amplificationsystem can further amplify signals to a higher level.

The bioconjugated polymer complexes described in FIGS. 6, 7, 8, 9, 10,14, 15, 16 and 17 can be used to create optically enhanced sandwichassays by directly integrating a light harvesting conjugated polymerinto commonly utilized recognition elements. The benefits of theconjugated polymer conjugated structures can also be applied directly tothe primary target recognition elements without the need for successiverecognition elements. For example, a primary antibody can be directlyconjugated to polymer-dye complex such as shown in FIG. 14. Such acomplex can be used to directly probe the presence of a targetbiomolecule.

Polynucleotides

Amplified target polynucleotides may be subjected to post amplificationtreatments. For example, in some cases, it may be desirable to fragmentthe target polynucleotide prior to hybridization in order to providesegments which are more readily accessible. Fragmentation of the nucleicacids can be carried out by any method producing fragments of a sizeuseful in the assay being performed; suitable physical, chemical andenzymatic methods are known in the art.

An amplification reaction can be performed under conditions which allowthe sensor polynucleotide to hybridize to the amplification productduring at least part of an amplification cycle. When the assay isperformed in this manner, real-time detection of this hybridizationevent can take place by monitoring for light emission duringamplification.

Real time PCR product analysis (and related real timereverse-transcription PCR) provides a well-known technique for real timePCR monitoring that has been used in a variety of contexts, which can beadapted for use with the methods described herein (see, Laurendeau etal. (1999) “TaqMan PCR-based gene dosage assay for predictive testing inindividuals from a cancer family with INK4 locus haploinsufficiency”Clin Chem 45(7):982-6; Laurendeau et al. (1999) “Quantitation of MYCgene expression in sporadic breast tumors with a real-time reversetranscription-PCR assay” Clin Chem 59(12):2759-65; and Kreuzer et al.(1999) “LightCycler technology for the quantitation of bcr/abl fusiontranscripts” Cancer Research 59(13):3171-4, all of which areincorporated by reference).

Samples

In principle, a sample can be any material suspected of containing atarget biomolecule (e.g., antibody, protein, affinity ligand, peptide,nucleic acid and the like) capable of causing excitation of a conjugatedpolymer complex. In some embodiments, the sample can be any source ofbiological material which comprises biomolecules that can be obtainedfrom a living organism directly or indirectly, including cells, tissueor fluid, and the deposits left by that organism, including viruses,mycoplasma, and fossils. The sample may comprise a target biomoleculeprepared through synthetic means, in whole or in part. Typically, thesample is obtained as or dispersed in a predominantly aqueous medium.Nonlimiting examples of the sample include blood, urine, semen, milk,sputum, mucus, a buccal swab, a vaginal swab, a rectal swab, anaspirate, a needle biopsy, a section of tissue obtained for example bysurgery or autopsy, plasma, serum, spinal fluid, lymph fluid, theexternal secretions of the skin, respiratory, intestinal, andgenitourinary tracts, tears, saliva, tumors, organs, samples of in vitrocell culture constituents (including but not limited to conditionedmedium resulting from the growth of cells in cell culture medium,putatively virally infected cells, recombinant cells, and cellcomponents), and a recombinant library comprising polynucleotidesequences.

The sample can be a positive control sample which is known to containthe target biomolecule or a surrogate therefore. A negative controlsample can also be used which, although not expected to contain thetarget biomolecule, is suspected of containing it (via contamination ofone or more of the reagents) or another component capable of producing afalse positive, and is tested in order to confirm the lack ofcontamination by the target biomolecule of the reagents used in a givenassay, as well as to determine whether a given set of assay conditionsproduces false positives (a positive signal even in the absence oftarget biomolecule in the sample).

The sample can be diluted, dissolved, suspended, extracted or otherwisetreated to solubilize and/or purify any target polynucleotide present orto render it accessible to reagents which are used in an amplificationscheme or to detection reagents. Where the sample contains cells, thecells can be lysed or permeabilized to release the polynucleotideswithin the cells. One step permeabilization buffers can be used to lysecells which allow further steps to be performed directly after lysis,for example a polymerase chain reaction.

Organic Dyes

Organic dyes include signaling chromophores and fluorophores. In someembodiments, a signaling chromophore or fluorophore may be employed, forexample to receive energy transferred from an excited state of anoptically active unit, or to exchange energy with a labeled probe, or inmultiple energy transfer schemes. Fluorophores useful in the inventionsdescribed herein include any substance which can absorb energy of anappropriate wavelength and emit or transfer energy. For multiplexedassays, a plurality of different fluorophores can be used withdetectably different emission spectra. Typical fluorophores includefluorescent dyes, semiconductor nanocrystals, lanthanide chelates, andfluorescent proteins.

Exemplary fluorescent dyes include fluorescein, 6-FAM, rhodamine, TexasRed, tetramethylrhodamine, a carboxyrhodamine, carboxyrhodamine 6G,carboxyrhodol, carboxyrhodamine 110, Cascade Blue, Cascade Yellow,coumarin, Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®, Cy-Chrome, DyLight 350,DyLight 405, DyLight 488, DyLight 549, DyLight 594, DyLight 633, DyLight649, DyLight 680, DyLight 750, DyLight 800, phycoerythrin, PerCP(peridinin chlorophyll-a Protein), PerCP-Cy5.5, JOE(6-carboxy-4′,5′-dichloro-2′,7′-dimelhoxyfluorescein), NED, ROX(5-(and-6)-carboxy-X-rhodamine), HEX, Lucifer Yellow, Marina Blue,Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Fluor® 350,Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546,Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 647,Alexa Fluor® 660, Alexa Fluor® 680, 7-amino-4-methylcoumarin-3-aceticacid, BODIPY® FL, BODIPY® FL-Br₂, BODIPY® 530/550, BODIPY® 558/568,BODIPY® 564/570, BODIPY® 576/589, BODIPY® 581/591, BODIPY® 630/650,BODIPY® 650/665, BODPY® R6G, BODIPY® TMR, BODIPY® TR, conjugatesthereof, and combinations thereof. Exemplary lanthanide chelates includeeuropium chelates, terbium chelates and samarium chelates.

A wide variety of fluorescent semiconductor nanocrystals (“SCNCs”) areknown in the art; methods of producing and utilizing semiconductornanocrystals are described in: PCT Publ. No. WO 99/26299 published May27, 1999, inventors Bawendi et al.; U.S. Pat. No. 5,990,479 issued Nov.23, 1999 to Weiss et al.; and Bruchez et al., Science 281:2013, 1998.Semiconductor nanocrystals can be obtained with very narrow emissionbands with well-defined peak emission wavelengths, allowing for a largenumber of different SCNCs to be used as signaling chromophores in thesame assay, optionally in combination with other non-SCNC types ofsignaling chromophores.

Exemplary polynucleotide-specific dyes include acridine orange, acridinehomodimer, actinomycin D, 7-aminoactmomycin D (7-AAD),9-amino-6-chlor-2-methoxyacridine (ACMA), BOBO™-1 iodide (462/481),BOBO™-3 iodide (570/602), BO-PRO™-1 iodide (462/481), BO-PRO™-3 iodide(575/599), 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI),4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI),4′,6-diamidino-2-phenylindole, dilactate (DAPI, dilactate),dihydroethidium (hydroethidine), dihydroethidium (hydroethidine),dihydroethidium (hydroethidine), ethidium bromide, ethidium diazidechloride, ethidium homodimer-1 (EthD-1), ethidium homodimer-2 (EthD-2),ethidium monoazide bromide (EMA), hexidium iodide, Hoechst 33258,Hoechst 33342, Hoechst 34580, Hoechst S769121, hydroxystilbamidine,methanesulfonate, JOJO™-1 iodide (529/545), JO-PRO™-1 iodide (530/546),LOLO™-1 iodide (565/579), LO-PRO™-1 iodide (567/580), NeuroTrace™435/455, NeuroTrace™ 500/525, NeuroTrace™ 515/535, NeuroTrace™ 530/615,NeuroTrace™ 640/660, OliGreen, PicoGreen® ssDNA, PicoGreen® dsDNA,POPO™-1 iodide (434/456), POPO™-3 iodide (534/570), PO-PRO™-1 iodide(435/455), PO-PRO™-3 iodide (539/567), propidium iodide, RiboGreen®,SlowFade®, SlowFade® Light, SYBR® Green I, SYBR® Green II, SYBR® Gold,SYBR® 101, SYBR® 102, SYBR® 103, SYBR® DX, TO-PRO®-1, TO-PRO®-3,TO-PRO®-5, TOTO®-1, TOTO®-3, YO-PRO®-1 (oxazole yellow), YO-PRO®-3,YOYO®-1, YOYO®-3, TO, SYTOX® Blue, SYTOX® Green, SYTOX® Orange, SYTO® 9,SYTO® BC, SYTO® 40, SYTO® 41, SYTO® 42, SYTO® 43, SYTO® 44, SYTO® 45,SYTO® Blue, SYTO® 11, SYTO® 12, SYTO® 13, SYTO® 14, SYTO® 15, SYTO® 16,SYTO® 20, SYTO® 21, SYTO® 22, SYTO® 23, SYTO® 24, SYTO® 25, SYTO® Green,SYTO® 80, SYTO® 81, SYTO® 82, SYTO® 83, SYTO® 84, SYTO® 85, SYTO®Orange, SYTO® 17, SYTO® 59, SYTO® 60, SYTO® 61, SYTO® 62, SYTO® 63,SYTO® 64, SYTO® Red, netropsin, distamycin, acridine orange,3,4-benzopyrene, thiazole orange, TOMEHE, daunomycin, acridine,pentyl-TOTAB, and butyl-TOTIN. Asymmetric cyanine dyes may be used asthe polynucleotide-specific dye. Other dyes of interest include thosedescribed by Geierstanger, B. H. and Wemmer, D. E., Annu. Rev. Vioshys.Biomol. Struct. 1995, 24, 463-493, by Larson, C. J. and Verdine, G. L.,Bioorganic Chemistry: Nucleic Acids, Hecht, S. M., Ed., OxfordUniversity Press: New York, 1996; pp 324-346, and by Glumoff, T. andGoldman, A. Nucleic Acids in Chemistry and Biology, 2^(nd) ed.,Blackburn, G. M. and Gait, M. J., Eds., Oxford University Press: Oxford,1996, pp 375-441. The polynucleotide-specific dye may be anintercalating dye, and may be specific for double-strandedpolynucleotides.

The term “fluorescent protein” includes types of protein known to absorband emit light. One of the more commonly used classes of such materialsare phycobiliproteins. Examples include but are not limited tophycoerythrin (PE and R-PE), allophycocyanin (APC) and PerCP. Otherclasses include green fluorescent protein and related versions.

The term “green fluorescent protein” refers to both native Aequoreagreen fluorescent protein and mutated versions that have been identifiedas exhibiting altered fluorescence characteristics, including alteredexcitation and emission maxima, as well as excitation and emissionspectra of different shapes (Delagrave, S. et al. (1995) Bio/Technology13:151-154; Heim, R. et al. (1994) Proc. Natl. Acad. Sci. USA91:12501-12504; Heim, R. et al. (1995) Nature 373:663-664). Delgrave etal. isolated mutants of cloned Aequorea victoria GFP that hadred-shifted excitation spectra. Bio/Technology 13:151-154 (1995). Heim,R. et al. reported a mutant (Tyr66 to His) having a blue fluorescence(Proc. Natl. Acad. Sci. (1994) USA 91:12501-12504).

Substrates

In some embodiments, an assay component can be located upon a substrate.The substrate can comprise a wide range of material, either biological,nonbiological, organic, inorganic, or a combination of any of these. Forexample, the substrate may be a polymerized Langmuir Blodgett film,functionalized glass, Si, Ge, GaAs, GaP, SiO₂, SiN₄, modified silicon,or any one of a wide variety of gels or polymers such as(poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene,cross-linked polystyrene, polyacrylic, polylactic acid, polyglycolicacid, poly(lactide coglycolide), polyanhydrides, poly(methylmethacrylate), poly(ethylene-co-vinyl acetate), polysiloxanes, polymericsilica, latexes, dextran polymers, epoxies, polycarbonates, orcombinations thereof. Conducting polymers and photoconductive materialscan be used.

Substrates can be planar crystalline substrates such as silica basedsubstrates (e.g. glass, quartz, or the like), or crystalline substratesused in, e.g., the semiconductor and microprocessor industries, such assilicon, gallium arsenide, indium doped GaN and the like, and includessemiconductor nanocrystals.

The substrate can take the form of a photodiode, an optoelectronicsensor such as an optoelectronic semiconductor chip or optoelectronicthin-film semiconductor, or a biochip. The location(s) of probe(s) onthe substrate can be addressable; this can be done in highly denseformats, and the location(s) can be microaddressable or nanoaddressable.

Silica aerogels can also be used as substrates, and can be prepared bymethods known in the art. Aerogel substrates may be used as freestanding substrates or as a surface coating for another substratematerial.

The substrate can take any form and typically is a plate, slide, bead,pellet, disk, particle, microparticle, nanoparticle, strand,precipitate, optionally porous gel, sheets, tube, sphere, container,capillary, pad, slice, film, chip, multiwell plate or dish, opticalfiber, etc. The substrate can be any form that is rigid or semi-rigid.The substrate may contain raised or depressed regions on which an assaycomponent is located. The surface of the substrate can be etched usingwell known techniques to provide for desired surface features, forexample trenches, v-grooves, mesa structures, or the like.

Surfaces on the substrate can be composed of the same material as thesubstrate or can be made from a different material, and can be coupledto the substrate by chemical or physical means. Such coupled surfacesmay be composed of any of a wide variety of materials, for example,polymers, plastics, resins, polysaccharides, silica or silica-basedmaterials, carbon, metals, inorganic glasses, membranes, or any of theabove-listed substrate materials. The surface can be opticallytransparent and can have surface Si—OH functionalities, such as thosefound on silica surfaces.

The substrate and/or its optional surface can be chosen to provideappropriate characteristics for the synthetic and/or detection methodsused. The substrate and/or surface can be transparent to allow theexposure of the substrate by light applied from multiple directions. Thesubstrate and/or surface may be provided with reflective “mirror”structures to increase the recovery of light.

The substrate and/or its surface is generally resistant to, or istreated to resist, the conditions to which it is to be exposed in use,and can be optionally treated to remove any resistant material afterexposure to such conditions.

Polynucleotide or polypeptide probes can be fabricated on or attached tothe substrate by any suitable method, for example the methods describedin U.S. Pat. No. 5,143,854, PCT Publ. No. WO 92/10092, U.S. patentapplication Ser. No. 07/624,120, filed Dec. 6, 1990 (now abandoned),Fodor et al., Science, 251: 767-777 (1991), and PCT Publ. No. WO90/15070). Techniques for the synthesis of these arrays using mechanicalsynthesis strategies are described in, e.g., PCT Publication No. WO93/09668 and U.S. Pat. No. 5,384,261.

Still further techniques include bead based techniques such as thosedescribed in PCT Appl. No. PCT/US93/04145 and pin based methods such asthose described in U.S. Pat. No. 5,288,514.

Additional flow channel or spotting methods applicable to attachment ofsensor polynucleotides or polypeptides to the substrate are described inU.S. patent application Ser. No. 07/980,523, filed Nov. 20, 1992, andU.S. Pat. No. 5,384,261. Reagents are delivered to the substrate byeither (1) flowing within a channel defined on predefined regions or (2)“spotting” on predefined regions. A protective coating such as ahydrophilic or hydrophobic coating (depending upon the nature of thesolvent) can be used over portions of the substrate to be protected,sometimes in combination with materials that facilitate wetting by thereactant solution in other regions. In this manner, the flowingsolutions are further prevented from passing outside of their designatedflow paths.

Typical dispensers include a micropipette optionally roboticallycontrolled, an ink-jet printer, a series of tubes, a manifold, an arrayof pipettes, or the like so that various reagents can be delivered tothe reaction regions sequentially or simultaneously.

The substrate or a region thereof may be encoded so that the identity ofthe sensor located in the substrate or region being queried may bedetermined. Any suitable coding scheme can be used, for example opticalcodes, RFID tags, magnetic codes, physical codes, fluorescent codes, andcombinations of codes.

Excitation and Detection

Any instrument that provides a wavelength that can excite the conjugatedpolymer complex and is shorter than the emission wavelength(s) to bedetected can be used for excitation. Commercially available devices canprovide suitable excitation wavelengths as well as suitable detectioncomponents.

Exemplary excitation sources include a broadband UV light source such asa deuterium lamp with an appropriate filter, the output of a white lightsource such as a xenon lamp or a deuterium lamp after passing through amonochromator to extract out the desired wavelengths, a continuous wave(cw) gas laser, a solid state diode laser, or any of the pulsed lasers.Emitted light can be detected through any suitable device or technique;many suitable approaches are known in the art. For example, afluorimeter or spectrophotometer may be used to detect whether the testsample emits light of a wavelength characteristic of the signalingchromophore upon excitation of the conjugated polymer.

Compositions of Matter

Also provided are compositions of matter of any of the moleculesdescribed herein in any of various forms. The conjugated polymers andcomplexes including conjugated polymers as described herein may beprovided in purified and/or isolated form. The conjugated polymers andcomplexes including conjugated polymers may be provided in eithercrystalline or amorphous form.

The conjugated polymers and complexes including conjugated polymers maybe provided in solution, which may be a predominantly aqueous solution,which may comprise one or more of the additional solution componentsdescribed herein, including without limitation additional solvents,buffers, biomolecules, polynucleotides, fluorophores, etc. In addition,a mixture of CPs in solution is also able to provide improved detectionsensitivity as compared to that for a single CP/dye system. Theconjugated polymers and complexes including conjugated polymers can bepresent in solution at a concentration at which a first emission fromthe first optically active units can be detected in the absence ofbiomolecule target or a biomolecule associated therewith. The solutionmay comprise additional components as described herein, includinglabeled probes such as fluorescently labeled antibodies orpolynucleotides, specific for a species or a class of biomolecule targetor a biomolecule associated therewith for the conjugated polymers andcomplexes including conjugated polymers.

The conjugated polymers and complexes including conjugated polymers maybe provided in the form of a film. The compositions of matter may beclaimed by any property described herein, including by proposedstructure, by method of synthesis, by absorption and/or emissionspectrum, by elemental analysis, by NMR spectra, or by any otherproperty or characteristic.

In some embodiments expression of a gene is detected in a sample. In afurther embodiment identification of a cell marker or cell type isdetected in a sample either in a flow cytometer, cell sorter,microscope, plate reader or fluorescence imager. In a further embodimentthe identification of cell type or marker is used in the diagnosis oflymphoma or other circulating cancers. In a further embodiment theidentification of cell type or marker is used in the diagnosis andmonitoring of HIV infection. In a further embodiment the identificationof cell type or marker is used to sort cells for therapeuticapplication. In a further embodiment, a measured result of detecting abiomolecule target or a biomolecule associated therewith can be used todiagnose a disease state of a patient. In yet another embodiment thedetection method of the invention can further include a method ofdiagnosing a disease state. In a related embodiment, the method ofdiagnosing a disease can include reviewing or analyzing data relating tothe presence of a biomolecule target or a biomolecule associatedtherewith and providing a conclusion to a patient, a health careprovider or a health care manager, the conclusion being based on thereview or analysis of data regarding a disease diagnosis. Reviewing oranalyzing such data can be facilitated using a computer or other digitaldevice and a network as described herein. It is envisioned thatinformation relating to such data can be transmitted over the network.

In practicing the methods of the present invention, many conventionaltechniques in molecular biology are optionally utilized. Thesetechniques are well known and are explained in, for example, Ausubel etal. (Eds.) Current Protocols in Molecular Biology, Volumes I, II, andIII, (1997), Ausubel et al. (Eds.), Short Protocols in MolecularBiology: A Compendium of Methods from Current Protocols in MolecularBiology, 5^(th) Ed., John Wiley & Sons, Inc. (2002), Sambrook et al.,Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring HarborLaboratory Press (2000), Innis et al. (Eds.) PCR Protocols: A Guide toMethods and Applications, Elsevier Science & Technology Books (1990),and Greg T. Hermanson, Bioconjugate Techniques, 2^(nd) Ed., AcademicPress, Inc. (2008) all of which are incorporated herein by reference.

FIG. 12 is a block diagram showing a representative example logic devicethrough which reviewing or analyzing data relating to the presentinvention can be achieved. Such data can be in relation to a disease,disorder or condition in a subject. FIG. 12 shows a computer system (ordigital device) 800 connected to an apparatus 820 for use with theconjugated polymers or conjugated polymers complexes 824 to, forexample, produce a result. The computer system 800 may be understood asa logical apparatus that can read instructions from media 811 and/ornetwork port 805, which can optionally be connected to server 809 havingfixed media 812. The system shown in FIG. 12 includes CPU 801, diskdrives 803, optional input devices such as keyboard 815 and/or mouse 816and optional monitor 807. Data communication can be achieved through theindicated communication medium to a server 809 at a local or a remotelocation. The communication medium can include any means of transmittingand/or receiving data. For example, the communication medium can be anetwork connection, a wireless connection or an internet connection. Itis envisioned that data relating to the present invention can betransmitted over such networks or connections.

In one embodiment, a computer-readable medium includes a medium suitablefor transmission of a result of an analysis of a biological sample. Themedium can include a result regarding a disease condition or state of asubject, wherein such a result is derived using the methods describedherein.

Kits

Kits comprising reagents useful for performing described methods arealso provided.

In some embodiments, a kit comprises reagents including conjugatedpolymers or conjugated polymers complexes, bioconjugates, for example,antibodies, nucleic acids, and other components as described herein.

The kit may optionally contain one or more of the following: one or morelabels that can be incorporated into conjugated polymers or conjugatedpolymers complexes; and one or more substrates which may or may notcontain an array, etc.

The components of a kit can be retained by a housing. Instructions forusing the kit to perform a described method can be provided with thehousing, and can be provided in any fixed medium. The instructions maybe located inside the housing or outside the housing, and may be printedon the interior or exterior of any surface forming the housing thatrenders the instructions legible. A kit may be in multiplex form fordetection of one or more different target biomolecules or biomoleculesassociated therewith.

As described herein and shown in FIG. 13, in certain embodiments a kit903 can include a container or housing 902 for housing variouscomponents. As shown in FIG. 13, and described herein, in one embodimenta kit 903 comprising one or more conjugated polymers or conjugatedpolymers complexes reagents 905, and optionally a substrate 900 isprovided. As shown in FIG. 13, and described herein, the kit 903 canoptionally include instructions 901. Other embodiments of the kit 903are envisioned wherein the components include various additionalfeatures described herein.

EXAMPLES

The following examples provide illustrative methods for making andtesting the effectiveness of the conjugated polymers described herein.These examples are provided for illustrative purposes only and not tolimit the scope of the claims provided herein. All of the methodsdisclosed and claimed herein can be made and executed without undueexperimentation in light of the present disclosure. It will be apparentto those of skill in the art that variations may be applied to themethods and in the steps or in the sequence of steps of the methoddescribed herein without departing from the concept, spirit and scope ofthe claims. All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope andconcept of the appended claims.

Example 1: Synthesis of a Polymer of Formula (I) Example 1a: Synthesisof Monomers,2,7-dibromo-9,9-di(2′,5′,8′,11′,14′,17′,23′,26′,29′,32′,35′-dodecaoxaoctatriacontan-38′-yl)fluorene(A) and9,9-di(2′,5′,8′,11′,14′,17′,20′,23′,26′,29′,32′,35′-dodecaoxaoctatriacontan-38′-yl)-2,7-di(4″,4″,5″,5″-tetramethyl-1″,3″,2″-dioxaborolanyl)fluorene(B) for Subsequent Polymerization

Step 1: 2,7-dibromo-9,9-di(3′-hydroxypropanyl)fluorene

2,7-dibromofluorene (9.72 g, 30 mmol), tetrabutylammonium bromide (300mg, 0.93 mmol), and DMSO (100 mL) were added to a 3-neck flask undernitrogen(g), followed by the addition of 50% NaOH (15 mL, 188 mmol) viasyringe. The mixture was heated to 80° C., and 3-bromopropanol (6.70 mL,77 mmol) was added dropwise via addition funnel, and the reactionmixture was stirred at 80° C. for another 2 hours. Upon completion, themixture was cooled to room temperature and quenched with water (250 mL).The aqueous layer was extracted with ethyl acetate (3 150 mL portions).The organic layers were combined, washed with water, then dried overMgSO₄, and filtered. The solvent was removed and the residual wasrecrystallized in chloroform to yield pale yellow needle crystals (9.20g, 70%).

Step 2: 2,7-dibromo-9,9-di(3′-methylbenzenesulfonatopropanyl)fluorene

2,7-dibromo-9,9-di(3′-hydroxypropanyl)fluorene (500 mg, 1.14 mmol) wasdissolved in dichloromethane (5 mL) at 0° C. under nitrogen(g). To themixture, added p-toluenesulfonyl chloride (650 mg, 3.40 mmol), followedby pyridine (0.39 mL, 4.77 mmol). Allowed reaction to stir at 0° C. andnaturally rise to room temperature over night. The reaction was quenchedwith water (15 mL). Removal of solvent by vacuo resulted solidsformation. Filtered off solids to yield white solids (758 mg, 89%).

Step 3:2,7-dibromo-9,9-di(2′,5′,8′,11′,14′,17′,23′,26′,29′,32′,35′-dodecaoxaoctatriacontan-38′-yl)fluorene(A)

mPEG11 alcohol (770 mg, 1.49 mmol) was dissolved in anhydrous THF (2 mL)at 0° C. under nitrogen. To the mixture, was added potassiumtert-butoxide (1.63 mmol, 1.63 mL, 1M in THF). After 10 min stirring,2,7-dibromo-9,9-di(3′-methylbenzenesulfonatopropanyl)fluorene (504 mg,0.673 mmol) in 10 mL of THF was added via a syringe. The mixture wasallowed to room temperature and stirred overnight. The reaction mixturewas diluted with THF. The insoluble inorganic salt was removed byfiltration. Concentration of the filtrate yielded crude product, whichwas purified by column chromatography (DCM-MeOH) to yield a colorlessoil (605 mg, 62.5%).

Step 4:9,9-di(2′,5′,8′,11′,14′,17′,20′,23′,26′,29′,32′,35′-dodecaoxaoctatriacontan-38′-yl)-2,7-di(4″,4″,5″,5″-tetramethyl-1″,3″,2″-dioxaborolanyl)fluorene(B)

2,7-dibromo-9,9-di(2′,5′,8′,11′,14′,17′,23′,26′,29′,32′,35′-dodecaoxaoctatriacontan-38′-yl)fluorene(1.510 g, 1.501 mmol), bis(pinacolato)diboron (800 mg, 3.15 mmol),potassium acetate (619 mg, 6.31 mmol), Pd(dppf)Cl₂[1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium(II)](51.5 mg,0.063 mmol) and DMSO (30 mL) were mixed under N₂. The mixture was heatedat 80° C. for 5.5 hour. Upon completion, the DMF was distilled and water(50 mL) was added. The product was extracted with DCM (3×40 mL). Theorganic layers were combined and concentrated. The crude product waspurified by column chromatography (DCM-MeOH) to give colorless oil(1.015 g, 63%).

Example 1b: Polymerization of Monomers (A) and (B)

2,7-dibromo-9,9-di(2′,5′,8′,11′,14′,17′,23′,26′,29′,32′,35′-dodecaoxaoctatriacontan-38′-yl)fluorene(A) (0.084 mmol, 120 mg),9,9-di(2′,5′,8′,11′,14′,17′,20′,23′,26′,29′,32′,35′-dodecaoxaoctatriacontan-38′-yl)-2,7-di(4″,4″,5″,5″-tetramethyl-1″,3″,2″-dioxaborolanyl)fluorene(B) (0.088 mmol, 135 mg), and palladium tetra(triphenylphosphine)(0.0035 mmol, 4 mg) were combined in a round bottom flask equipped witha stirbar. Next, 0.35 mL of 2M potassium carbonate (aq) and 1.9 mL oftetrahydrofuran were added and the flask is fitted with a vacuum adaptorand put on a Schlenk line. The mixture was degassed using 3freeze-pump-thaw cycles. The degassed mixture was heated to 80° C. undernitrogen with vigorous stirring for 18 hours. The reaction mixture wasthen cooled and the solvent removed with rotary evaporation. Theresulting semisolid was diluted with ca. 50 mL water and filteredthrough glass fiber filter paper. Ethanol was added to adjust thesolvent to 20% ethanol in water. Preparative gel permeationchromatography was performed with G-25 desalting medium to remove excesssalts from the polymer. Solvent in the fractions was removed with rotaryevaporation and 100 mg of poly [2,7{9,9-bis (2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane)fluorene] wascollected as an amber oil.

Example 2: Synthesis of Asymmetric Polymers of Formula (I) Via SuzukiCoupling Example 2a: Synthesis of Asymmetric Monomer,2-bromo-9,9-di(2′,5′,8′,11′,14′,17′,20′,23′,26′,29′,32′,35′-dodecaoxaoctatriacontan-38′-yl)-7-(4″,4″,5″,5″-tetramethyl-1″,3″,2″-dioxaborolan-2-yl)fluorene(C) for Subsequent Polymerization

Step 2: 2-dibromo-7-iodofluorene

2-bromofluorene (10.01 g, 40.84 mmol), acetic acid (170 mL), water (8mL), iodine (4.34 g, 17.20 mmol), potassium iodate (2.18 g, 10.19 mmol)and sulfuric acid (4 mL) were mixed under nitrogen. The resultingmixture was heated at 80° C. for 2 h and cooled to room temperature. Theformed precipitate which is the desired product was collected afterfiltration and acetic acid wash (13.68 g, 90%).

Step 2: 2-dibromo-9,9-di(3′hydroxypropanyl)-7-iodofluorene

2-dibromo-7-iodofluorene (2.186 g, 5.892 mmol), tetrabutylammoniumbromide (60 mg, 0.186 mmol), and DMSO (25 mL) were added to a 3-neckflask under nitrogen(g), followed by the addition of 50% NaOH (4 mL, 50mmol) via syringe. The mixture was heated to 80° C., and 3-bromopropanol(1.33 mL, 14.7 mmol) was added slowly, and the reaction was stirred at80° C. for another 1 hour. Upon completion, the mixture was cooled toroom temperature and quenched with water. The precipitate as crudeproduct was collected after filtration. The crude product was purifiedby column chromatography (eluant: hexane-ethylacetate) to give paleyellow solid (2.15 g, 75%).

Step 3:2-bromo-9,9-di(3′-hydroxypropanyl)-7-(4″,4″,5″,5″-tetramethyl-1″,3″,2″-dioxaborolan-2-yl)fluorene

2-dibromo-9,9-di(3′hydroxypropanyl)-7-iodofluorene (2.454 g, 5.037mmol), bis(pinacolato)diboron (1.407 g, 5.541 mmol), potassium acetate(1.483 g, 15.11 mmol), Pd(dppf)Cl₂ (123 mg, 0.15 mmol) and DMSO (25 mL)were mixed under N₂. The mixture was heated at 80° C. for 1.5 hour. Uponcompletion, the mixture was cooled to room temperature and quenched withwater (50 mL). The product was extracted with DCM (3×40 mL). The organiclayers were combined and concentrated. The crude product was purified bycolumn chromatography (eluant: hexane-ethylacetate) to give pale solid(2.09 g, 85%).

Step 4:2-bromo-9,9-di(3′-methanesulfanotopropanyl)-7-(4″,4″,5″5″-tetramethyl-1″,3″,2″-dioxaborolan-2-yl)fluorene

2-bromo-9,9-di(3′-hydroxypropanyl)-7-(4″,4″,5″,5″-tetramethyl-1″,3″,2″-dioxaborolan-2-yl)fluorene(2.280 g, 4.694 mmol) and p-toluenesulfonyl chloride (2.684 g, 14.08mmol) were dissolved in dichloromethane at room temperature under N₂.Triethylamine (3.95 mL, 28.2 mmol) was added slowly via syringe. Themixture was stirred at room temperature over night. The mixture was thenconcentrated and purified by column chromatography (Hexane-EtOAc) toyield pale solid (2.66 g, 72%).

Step 5:2-bromo-9,9-di(2′,5′,8′,11′,14′,17′,20′,23′,26′,29′,32′,35′-dodecaoxaoctatriacontan-38′-yl)-7-(4″,4″,5″,5″-tetramethyl-1″,3″,2″-dioxaborolan-2-yl)fluorine(C)

mPEG11 alcohol (3.331 g, 6.448 mmol) was dissolved in anhydrous THF (20mL) at 0° C. under nitrogen. To the mixture, was added potassiumtert-butoxide (7.74 mmol, 7.74 mL, 1M in THF). After 10 min stirring,2-bromo-9,9-di(3′-methanesulfanotopropanyl)-7-(4″,4″,5″,5″-tetramethyl-1″,3″,2″-dioxaborolan-2-yl)fluorine(2.052 g, 2.579 mmol) in 20 mL of anhydrous THF was added via a syringe.The mixture was allowed to room temperature and stirred overnight. Afterevaporation of THF, brine (50 mL) was added and crude product wasextracted with dichloromethane (3×40 mL). The combined organic layerswere concentrated and purified by column chromatography(DCM-isopropanol) to give colorless gel-like product (2.164 g, 57%).

Example 2b: Synthesis of an Asymmetric Polymer Via Suzuki CouplingPolymerization

Asymmetric polymers are synthesized using conditions similar topolymerization conditions as described in Example 1b.

Example 3: Synthesis of a Linker or Capping Unit Example 3a: Synthesisof Linker or Capping Unit, Tert-butyl4-(3,5-dibromophenoxy)butylcarbamate

Step 1: 4-(3,5-dibromophenoxy)butan-1-amine

1-(4′-phthalimidobutoxy)3,5-dibromobenzene (1.0 g, 2.20 mmol) wasdissolved in ethanol (45 mL) for 5 minutes under nitrogen. Hydrazinemonohydrate (610 mg, 12.1 mmol) was added and the reaction was refluxedat 80° C. for 2 hours. To the reaction aqueous 1M HCl (17.7 mL, 17.7mmol) was added and refluxed at 105° C. for another 2 hours. The aqueouslayer was extracted with dichloromethane (2×150 mL). The organic layerswere combined, washed with saturated NaHCO₃ (3×), water, and brine, thendried over MgSO₄, and filtered. Removal of solvent yielded a yellow oil(560 mg, 78%).

Step 2: Tert-butyl 4-(3,5-dibromophenoxy)butylcarbamate

4-(3,5-dibromophenoxy)butan-1-amine (397 mg, 1.23 mmol) was dissolved inanhydrous THF (24.6 mL) under nitrogen. Di-tert-butyl dicarbonate (423mL, 1.84 mmol) was added to the mixture and refluxed reaction at 40° C.for 2 hours. After extraction of the reaction with dichloromethane (2×50mL), the organic layers were combined, washed with saturated NaHCO₃,water, and brine, then dried over MgSO₄, and filtered. The solvent isremoved and the residue is purified by column chromatography (9:1,hexanes: EtOAc) to give a white solid (306 mg, 59%).

Example 3b: Synthesis of Linker or Capping Unit,Tert-butyl-4-(2,7-dibromo-9-methyl-9H-fluoren-9-yl)butylcarbamate

Step 1: 2,7-dibromo-9-methyl-9H-fluorene

2,7-dibromofluorene (30 g, 92.59 mmol) was dissolved in anhydrous THF(300 mL) under nitrogen and cooled to −78° C. To solution at −78° C.,added n-butyllithium (40.36 mL, 100.9 mmol) over 5 minutes and allowedreaction stir for another 10 minutes. To reaction, then add methyliodide (6.29 mL, 100.9 mmol) and allowed reaction to stir at −78° C. for2.0 hours. The reaction was poured into a mixture of dichloromethane andwater. The organic layer was collected, and the water layer was furtherextracted with dichloromethane. Combined all organic layers and removedsolvent via vacuo. The crude material was triturated with hexanes andfiltered using Buchner funnel to give white solids (22 g, 70%). ¹H NMR(500 MHz, CDCl₃): δ=7.62 (s, 2H), 7.56-7.58 (d, 2H), 7.48-7.50 (dd, 2H),3.90-3.94 (q, 1H), 1.49-1.51 (d, 3H).

Step 2:2-(4-(2,7-dibromo-9-methyl-9H-fluoren-9-yl)butyl)isoindoline-1,3-dione

2,7-dibromo-9-methyl-9H-fluorene (10.0 g, 29.58 mmol) was dissolved in50 mL DMSO under nitrogen. To mixture was added KOH (2.01 g, 35.79mmol), water (1.5 mL), N-(4-bromobutyl)phthalimide (9.93 g, 35.2 mmol),and stirred reaction at room temperature for 2.0 hours, then at 50° C.for 3.0 hours. The reaction was cooled to room temperature and dilutedwith dichloromethane. The organic layer was washed with brine (2×), andwater. Removal of solvent yield a solid, which was purified by columnchromatography (7:3, hexanes:EtOAc) to yield white solids (3.08 g, 20%).¹H NMR (500 MHz, CDCl₃): δ=7.81-7.83 (m, 2H), 7.68-7.71 (m, 2H),7.48-7.51 (m, 4H), 7.41-7.44 (dd, 2H), 3.46-3.49 (t, 2H), 2.00-2.04 (p,2H), 1.47-1.49 (m, 2H), 1.45 (s, 3H), 0.65-0.68 (m, 2H).

Step 3: 4-(2,7-dibromo-9-methyl-9H-fluoren-9-yl)butan-1-amine

2-(4-(2,7-dibromo-9-methyl-9H-fluoren-9-yl)butyl)isoindoline-1,3-dione(3.08, 5.71 mmol) was dissolved in ethanol (250 mL) under nitrogen. Tothe mixture was added hydrazine monohydrate (2.77 mL, 57.1 mmol), andthe reaction was refluxed at 80° C. for 3.0 hours. The reaction wascooled to room temperature, and added 1M HCl (˜100 mL). The mixture wasstirred for 30 minutes or until all solids were dissolved.Dichloromethane was added to the solution and the organic layer wasextracted with saturated NaHCO₃ three times, and washed with water. Theorganic layers were collected and removed solvent by vacuo to give anyellow oil (2.33 g, 100%). ¹H NMR (500 MHz, CD₂Cl₂): δ=7.57 (d, 2H),7.52 (d, 2H), 7.46-7.48 (dd, 2H), 2.39-2.42 (t, 2H), 1.95-1.98 (t, 2H),1.44 (s, 3H), 1.17-1.23 (m, 2H), 0.59-0.65 (m, 2H).

Step 4:tert-butyl-4-(2,7-dibromo-9-methyl-9H-fluoren-9-yl)butylcarbamate

4-(2,7-dibromo-9-methyl-9H-fluoren-9-yl)butan-1-amine (2.39 g, 5.84mmol) was dissolved in anhydrous THF (20 mL) under nitrogen. Tosolution, was added di-tert-butyl-dicarbonate (2.01 mL, 8.76 mmol), andthe reaction was stirred at 40° C. for 3 hours. The reaction was cooledto room temperature and concentrated via vacuo. Crude solids weretriturated with hexanes and filtered using buchner funnel to yield thedesired white solids (2.34 g, 79%). ¹H NMR (500 MHz, CDCl₃): δ=7.53 (d,2H), 7.45-7.47 (d, 4H), 4.30 (s, 1H), 2.88-2.90 (q, 2H), 1.93-1.96 (t,2H), 1.43 (s, 3H), 1.41 (s, 9H), 1.25-1.28 (m, 2H), 0.59-0.66 (m, 2H).

Example 4: Synthesis of a Linker or Capping Unit Example 4a: Synthesisof Tert-butyl 4-(4-bromophenoxy)butylcarbamate

Step 1: N(4-(4-bromophenoxy)butyl)phthalimide

Combined 4-bromophenol (4.64 g, 26.8 mmol), N-(4-bromobutylphthalimide)(6.30 g, 22.33 mmol), K₂CO₃ (11.09 g, 80.38 mmol), 18-crown-6 (265 mg,1.00 mmol), and acetone (100 mL), and refluxed reaction under nitrogenat 70° C. over night. The reaction was cooled to room temperature andremoved solvent by vacuum. The crude mixture was diluted withdichloromethane (200 mL) and washed with water (3×), then dried overMgSO₄, and filtered. Removal of solvent, followed by trituration withhexanes, and filtered using Buchner funnel to yield a white solid (6.03g, 71%).

Step 2: 4-(4-bromophenoxy)butan-1-amine

N(4-(4-bromophenoxy)butyl)phthalimide (6.01 g, 16.1 mmol) is dissolvedin ethanol (200 mL) under nitrogen, followed by the addition ofhydrazine monohydrate (7.8 mL, 161 mmol). The reaction was refluxed at80° C. for 2 hours. Once reaction completed (solids formed at the toplayer), cooled reaction to room temperature and neutralized with 1M HCl(50 mL). The mixture is allowed to stir until all solids are completelydissolved and diluted with dichloromethane (150 mL). The solution wasextracted with two portions of saturated NaHCO₃ (2×). The organic layerswere combined, washed with brine and water, then dried over MgSO₄, andfiltered. Removal of solvent yields a yellow oil (2.93 g, 75%).

Step 3: Tert-butyl 4-(4-bromophenoxy)butylcarbamate

4-(4-bromophenoxy)butan-1-amine (1.0 g, 4.09 mmol) was dissolved inanhydrous THF (20 mL) under nitrogen and stirred until solution ishomogenous. Di-tert-butyl-dicarbonate (1.34 g, 6.14 mmol) was added andthe reaction was stirred at 40° C. for 2 hours. The reaction wasquenched with water (30 mL) and stirred at room temperature for 1.0hour. The aqueous layer was extracted with ethyl acetate (50 mL×2). Theorganic layers were combined, washed with saturated NaHCO₃, water, andbrine, then dried over MgSO₄, and filtered. Removal of solvent yield asolid, which was purified by column chromatography (9:1, hexanes:EtOAc)to yield white solids (1.0 g, 71%).

Example 4b: Synthesis of tert-butyl 4-(4-bromophenyl)butanoate

Allowed tert-butanol to melt and added 20 mL to round bottom flask. Tothe solution, added di-tert-butyl-dicarbonate (1.79 g, 8.22 mmol) and4-(4-bromophenyl)butyric acid (1.0 g, 4.11 mmol). To reaction, thenadded DMAP (150.7 mg, 1.23 mmol) and stirred reaction at roomtemperature over night. The reaction was concentrated via vacuo, andre-diluted in ethyl acetate. The organic layer was washed with 1M HCl,brine, and water. After removal of solvent, the crude solids werepurified via column chromatography (20:1, hexanes:EtOAc) to give thedesired product (570 mg, 46%), which is a clear oil. ¹H NMR (500 MHz,CD₂Cl₂): δ=7.39-7.41 (d, 2H), 7.03-7.09 (d, 2H), 2.57-2.60 (t, 2H),2.18-2.21 (t, 2H), 1.83-1.186 (p, 2H), 1.42 (s, 9H).

Example 4c: Synthesis of4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)butanoic acid

Combined 4-(4-bromophenyl)butyric acid (10 g, 41.13 mmol),bis(pinacolato)diboron (15.67 g, 61.70 mmol), potassium acetate (12.1 g,123.4 mmol), and DMSO (100 mL), and purged mixture with nitrogen for 10minutes at room temperature. To reaction under nitrogen, addedPd(dppf)Cl₂ and purged reaction again with nitrogen for another 20minutes at room temperature. The reaction was then refluxed at 80° C.over night. After cooling to room temperature, the reaction was quenchedwith water and stirred for 1.0 hour. The solids formed were filteredusing Buchner funnel. The crude solids were purified via columnchromatography (8.5:1.5, hexanes:EtOAc). The desired fractions werecollected and concentrated via vacuo, and triturated with hexanes andfiltered to give the desired white solids (6.7 g, 56%).

Example 5: Synthesis of Linker or Capping Unit, Tert-butyl4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)butylcarbamate

Combined tert-butyl 4-(4-bromophenoxy)butylcarbamate from Example 4a(500 mg, 1.45 mmol), potassium acetate (428 mg, 4.36 mmol),bis(pinacolato)diboron (737 mg, 2.90 mmol) and DMSO (12 mL), and purgedmixture with nitrogen for 10 minutes at room temperature. To mixture wasadded Pd(dppf)Cl₂ (59.3 mg, 0.07 mmol) and continued to stir solution atroom temperature under nitrogen for another 20 minutes. After refluxingat 80° C. for 3 hours, the reaction was cooled to room temperature andquenched with water (30 mL). The aqueous layer was extracted withdichloromethane (50 mL×2). The organic layers were combined, washed withbrine, then dried over MgSO₄, and filtered. Removal of solvent yield adark brown oil, which was purified by column chromatography (9:1,hexanes:EtOAc) to yield a light yellow oil (539 mg, 95%).

Example 6: Synthesis of Linker or Capping Unit with Long OligoetherSpacer Between Arylhalide Phenyl and FMOC Protected Primary Amine

4-(4-bromophenoxy)butan-1-amine+oligoether-FMOC+N,N′-dicyclohexylcarbodiimide(DCC)

(9H-fluoren-9-yl)methyl80-(4-bromophenoxy)-75-oxo-3,6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51,54,57,60,63,66,69,72-tetracosaoxa-76-azaoctacontylcarbamate.4-(4-bromophenoxy)butan-1-amine (21.5 mg, 0.09 mmol),1-(9H-fluoren-9-yl)-3-oxo-2,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,58,61,64,67,70,73,76-pentacosaoxa-4-azanonaheptacontan-79-oicacid (100 mg, 0.073 mmol), and N,N′-dimethylaminopyridine (5.4 mg, 0.044mmol) were combined in a round bottom flask flushed with nitrogen andcharged with a Teflon stirbar. Next 5 mL of anhydrous dichloromethanewas added via syringe. N,N-Dicyclohexylcarbodiimide (23 mg, 0.11 mmol)is transferred to a second flask flushed with nitrogen and charged witha stirbar and 5 mL of anhydrous dichloromethane is added via syringe.While stirring the first solution, add the dicyclohexylcarbodiimidesolution slowly, dropwise. The reaction is then allowed to proceedovernight. The following day solids from the reaction were filtered offand the filtrate was concentrated onto silica. Column chromatography inmethanol and dichloromethane gave a clear thick oil (83.3 mg, 71%yield).

Example 7: Synthesis of Polymer,Poly[2,7{9,9-bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane)fluorene}-co-3,5-phenylbut-1′-oxy-4″-amine],with an Internal Linking Site

The incorporation of internal conjugation sites into conjugated polymerbackbones is described in U.S. application Ser. No. 11/868,870, filedOct. 8, 2007 and published as U.S. Application No. 2008/0293164, whichapplication is herein incorporated by reference in its entirety.Provided is a modified synthesis based on the protocol.

2,7-dibromo-9,9-di(2′,5′,8′,11′,14′,17′,23′,26′,29′,32′,35′-dodecaoxaoctatriacontan-38′-yl)fluorene(0.084 mmol, 120 mg),9,9-di(2′,5′,8′,11′,14′,17′,20′,23′,26′,29′,32′,35′-dodecaoxaoctatriacontan-38′-yl)-2,7-di(4″,4″,5″,5″-tetramethyl-1″,3″,2″-dioxaborolanyl)fluorene(0.088 mmol, 135 mg), tert-butyl-4-(3,5-dibromophenoxy)butylcarbamate(0.0044 mmol, 2.0 mg), and palladium tetra(triphenylphosphine) (0.0035mmol, 4 mg) are combined in a round bottom flask equipped with astirbar. Next, 0.35 mL of 2M potassium carbonate (aq) and 1.9 mL oftetrahydrofuran are added and the flask is fitted with a vacuum adaptorand put on a Schlenk line. The mixture is degassed using 3freeze-pump-thaw cycles. The degassed mixture is heated to 80 C undernitrogen with vigorous stirring for 18 hours. The reaction mixture isthen cooled and the solvent is removed with rotary evaporation. Next, 4mL of 4 M HCl in dioxane is added and the mixture is stirred for no lessthan 4 hours. The solution is neutralized with 2M potassium carbonatesolution. The bulk of the solvent is again removed with rotaryevaporation. The resulting semisolid is diluted with ca. 50 mL water andfiltered through glass fiber filter paper. Ethanol is added to adjustthe solvent to 20% ethanol in water. Preparative gel permeationchromatography is performed with G-25 desalting medium to remove excesssalts from the polymer. Solvent in the fractions is removed with rotaryevaporation and 100 mg of poly[2,7{9,9-bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane)fluorene}-co-3,5-tert-butyl-4-(4-bromophenoxy)amine]is collected as an amber oil.

Example 8: Synthesis of Phenylene Vinylene Co-Polymer with an InternalLinking Site

A modified synthesis similar to that described in Examples 7 and 15.

Example 9: Synthesis of Polymer with Exclusively Terminal Amine CappingUnits

2,7-(Poly[2,7{9,9-bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane)fluorene])-diphen-4-oxybutyl-4′-amine

2,7-dibromo-9,9-di(2′,5′,8′,11′,14′,17′,23′,26′,29′,32′,35′-dodecaoxaoctatriacontan-38′-yl)fluorene(0.163 mmol, 235 mg),9,9-di(2′,5′,8′,11′,14′,17′,20′,23′,26′,29′,32′,35′-dodecaoxaoctatriacontan-38′-yl)-2,7-di(4″,4″,5″,5″-tetramethyl-1″,3″,2″-dioxaborolanyl)fluorene(0.163 mmol, 250 mg), and palladium tetra(triphenylphoshine) (0.0065,7.5 mg) are combined in a round bottom flask equipped with a stirbar.Next, 0.75 mL of 2M potassium carbonate (aq) and 3 mL of tetrahydrofuranare added and the flask is fitted with a vacuum adaptor. The reactionmixture is put on a Schenk line and is degassed with threefreeze-pump-thaw cycles and then heated to 80 C under nitrogen withvigorous stirring for 18 hours. A solution of tert-butyl4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)butylcarbamate(0.064 mmol, 25 mg) in 0.5 mL tetrahydrofuran is degassed with threefreeze-pump-thaw cycles and then added to the polymerization reactionvia cannula under excess nitrogen pressure. The reaction is allowed tocontinue for an additional 4 hours at 80 C with stirring. Next, asolution of tert-butyl 4-(4-bromophenoxy)butylcarbamate (0.192 mmol, 66mg) in 0.5 mL of THF is degassed with three freeze-pump-thaw cycles andthen added to the polymerization reaction via cannula under excessnitrogen pressure. The reaction was allowed to proceed overnight. Thereaction mixture was allowed to cool and solvent was removed with rotaryevaporation. A 4 mL portion of 4M HCl in dioxane was added to theresidue and stirred for a minimum of 4 hours. The solution wasneutralized with 2 M potassium carbonate (aq) and then the solvent wasremoved under vacuum. The resulting residue was diluted to ˜30 mL with20% ethanol in water and filtered. Preparative gel permeationchromatography is performed with G-25 desalting medium to remove excesssalts from the polymer. Solvent in the fractions is removed with rotaryevaporation and 337 mg of polymer is collected as an amber oil.

The order of end linker addition (aryl hylide or boronic ester/acid) canbe reversed. Similar processes can be used to add alternative linkers orend capping units.

Example 10: Synthesis of Polymer, 2-(Poly[2,7{9,9-bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane)fluorene])-phen-4-oxybutyl-4′-amine,Statistically Enriched in Chains with a Single Terminal Amine CappingUnit

2,7-dibromo-9,9-di(2′,5′,8′,11′,14′,17′,23′,26′,29′,32′,35′-dodecaoxaoctatriacontan-38′-yl)fluorene(0.163 mmol, 235 mg),9,9-di(2′,5′,8′,11′,14′,17′,20′,23′,26′,29′,32′,35′-dodecaoxaoctatriacontan-38′-yl)-2,7-di(4″,4″,5″,5″-tetramethyl-1″,3″,2″-dioxaborolanyl)fluorene(0.163 mmol, 250 mg), and palladium tetra(triphenylphoshine) (0.0065,7.5 mg) are combined in a round bottom flask equipped with a stirbar.Next, 0.75 mL of 2M potassium carbonate (aq) and 3 mL of tetrahydrofuranare added and the flask is fitted with a vacuum adaptor. The reactionmixture is put on a Schenk line and is degassed with threefreeze-pump-thaw cycles and then heated to 80 C under nitrogen withvigorous stirring for 18 hours. A solution of tert-butyl4-(4-bromophenoxy)butylcarbamate (0.049 mmol, 17 mg) in 0.5 mLtetrahydrofuran is degassed with three freeze-pump-thaw cycles and thenadded to the polymerization reaction via cannula under excess nitrogenpressure. The reaction is allowed to continue for an additional 4 hoursat 80 C with stirring. Next, a solution of phenylboronic acid (0.150mmol, 18 mg) in 0.5 mL of THF is degassed with three freeze-pump-thawcycles and then added to the polymerization reaction via cannula underexcess nitrogen pressure. The reaction was allowed to proceed overnight.The reaction mixture was allowed to cool and solvent was removed withrotary evaporation. A 4 mL portion of 4M HCl in dioxanewas added to theresidue and stirred for a at least 4 hours. The solution was neutralizedwith 2 M potassium carbonate (aq) and then the solvent was removed undervacuum. The resulting residue was diluted to ˜30 mL with 20% ethanol inwater and filtered. Preparative gel permeation chromatography isperformed with G-25 desalting medium to remove excess salts from thepolymer. Solvent in the fractions is removed with rotary evaporation and315 mg of polymer is collected as an amber oil. Resulting polymerscontain chains with an enriched fraction of chains with one amine linkerplus chains with 2 linkers and no linkers.

Example 11: Synthesis of Polymer Statistically Enriched in Chains with aSingle Terminal Capping Unit with a Long Oligoether Spacer (24 Repeats)Between the Polymer Chain and the Primary Amine Linking Group

2-(Poly[2,7{9,9-bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane)fluorene])-phen-4-oxybutyl-4′-amine.2,7-dibromo-9,9-di(2′,5′,8′,11′,14′,17′,23′,26′,29′,32′,35′-dodecaoxaoctatriacontan-38′-yl)fluorene(0.163 mmol, 235 mg),9,9-di(2′,5′,8′,11′,14′,17′,20′,23′,26′,29′,32′,35′-dodecaoxaoctatriacontan-38′-yl)-2,7-di(4″,4″,5″,5″-tetramethyl-1″,3″,2″-dioxaborolanyl)fluorene(0.163 mmol, 250 mg), and palladium tetra(triphenylphoshine) (0.0065,7.5 mg) are combined in a round bottom flask equipped with a stirbar.Next, 0.75 mL of 2M potassium carbonate (aq) and 3 mL of tetrahydrofuranare added and the flask is fitted with a vacuum adaptor. The reactionmixture is put on a Schenk line and is degassed with threefreeze-pump-thaw cycles and then heated to 80 C under nitrogen withvigorous stirring for 18 hours. A solution of tert-butyl4-(4-bromophenoxy)butylcarbamate (0.049 mmol, 17 mg) in 0.5 mLtetrahydrofuran is degassed with three freeze-pump-thaw cycles and thenadded to the polymerization reaction via cannula under excess nitrogenpressure. The reaction is allowed to continue for an additional 4 hoursat 80 C with stirring. Next, a solution of phenylboronic acid (0.150mmol, 18 mg) in 0.5 mL of THF is degassed with three freeze-pump-thawcycles and then added to the polymerization reaction via cannula underexcess nitrogen pressure. The reaction was allowed to proceed overnight.The reaction mixture was allowed to cool and solvent was removed withrotary evaporation. A 4 mL portion of 4M HCl in dioxanewas added to theresidue and stirred for a minimum of 4 hours. The solution wasneutralized with 2 M potassium carbonate (aq) and then the solvent wasremoved under vacuum. The resulting residue was diluted to ˜30 mL with20% ethanol in water and filtered. Preparative gel permeationchromatography is performed with G-25 desalting medium to remove excesssalts from the polymer. Solvent in the fractions is removed with rotaryevaporation and 315 mg of polymer is collected as an amber oil.

Example 12: Synthesis of an Asymmetric Polymer with a TerminalCarboxylic Capping Unit Added During Polymerization Reaction

The linking monomer is added during the polymerization reaction asdescribed in Examples 9, 10 and 11. The carboxylic acid group can laterbe converted to an activated ester such as N-hydroxysuccinimidyl as isdescribed in Example 29.

Example 13: Synthesis of an Asymmetric Polymer with a TerminalCarboxylic Acid Capping Unit Added Post Polymerization

The linking monomer is added after the polymerization reaction iscompleted and polymer purified. Linker addition is done under similarreaction conditions as those described in Examples 9, 10 and 11. Thecarboxylic acid group can later be converted to an activated ester suchas N-hydroxysuccinimidyl as is described in Example 29.

Example 14: Synthesis of an Polymer with Branched PEG Groups Example14a: Synthesis of Monomers, (D) and (E) for Subsequent Polymerization

Step 1: 2,7-dibromo-9,9-bis(3,5-dimethoxybenzyl)fluorene

2,7-dibromofluorene (4.16 g, 12.8 mmol) and tetrabutylammonium bromide(362 mg, 1.12 mmol) were added to a round bottom flask charged with aTeflon stirbar. Next, 60 mL of dimethylsulfoxide was added to the flaskand the mixture was stirred for 5 minutes. A portion of 50% NaOH aqueoussolution (5.2 mL) was added followed immediately by 3,5-dimethoxybenzylbromide (7.14 g, 31 mmol). Over the course of 2 hours the solutionchanges color from orange to blue. The reaction is stirred overnight.The resulting mixture is slowly poured into 200 mL of water and thenextracted with three 100 mL portions of dichloromethane. The organiclayers are combined and dried over magnesium sulfate and then filtered.The crude product is purified by column chromatography using hexanes anddichloromethane as eluent to give a pale yellow solid (6.63 g, 79%yield).

Step 2: 2,7-dibromo-9,9-bis(3,5-dihydroxybenzyl)-9H-fluorene

2,7-dibromo-9,9-bis(3,5-dimethoxybenzyl)-9H-fluorene (1.3 g, 2.08 mmol)was added to a round bottom flask charged with a stirbar and equippedwith a rubber septum. The flask is purged with nitrogen for 10 min.Anhydrous dichloromethane (20 mL) is transferred to the flask viacannula and the mixture is stirred until the solids are completelydissolved. The solution is then cooled with a dry ice/acetone bath for10 minutes. BBr₃ (6.1 mL, 63.3 mmol) is added dropwise via cannula withconstant stirring. The bath is allowed to warm to room temperature andthe mixture is stirred overnight. The reaction is quenched with the slowaddition of 125 mL of water. The solution is then extracted with 3portions of ethyl acetate (50 mL). The organic layer is dried overMgSO₃, filtered, and dried onto silica. Flash chromatography of thecrude using ethyl acetate in dichloromethane gives an off-whitecrystalline solid (800 mg, 68% yield).

Step 3:2,7-dibromo-9,9-bis(3,5-(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yl)benzyl)-9H-fluorene (D)

2,7-dibromo-9,9-bis(3,5-dihydroxybenzyl)-9H-fluorene (537 mg, 0.945mmol), 2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yl4-methylbenzenesulfonate (2.788 g, 4.156 mmol), potassium carbonate(1.57 g, 11.34 mmol) and acetone (80 mL) are transferred to a roundbottom flask charged with a Teflon stirbar and equipped with a refluxcondenser. The mixture is refluxed with constant stirring overnight. Themixture is then allowed to cool to room temperature and the acetone isremoved under vacuum. After extracting with 3 portions ofdichloromethane, the organic layer is dried over MgSO₄, filtered, andthe filtrate is concentrated onto silica. Column chromatography usingmethanol and dichloromethane affords the product as a slightly coloredthick oil (1.69 g, 70% yield).

Step 4:2,7-di(4″,4″,5″,5″-tetramethyl-1″,3″,2″-dioxaborolanyl)-9,9-bis(3,5-(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yl)benzyl)-9H-fluorene (E)

Monomer (E) is synthesized using conditions similar to conditions asdescribed in Example 1.

Example 14b: Polymerization of (D) and (E)

Polymerization of (D) and (E) are polymerized using conditions similarto polymerization conditions as described in Example 1b.

Example 15: Synthesis of a Neutral Base Phenylene Vinylene Co-Polymer

2,7-dibromo-9,9-di(2′,5′,8′,11′,14′,17′,23′,26′,29′,32′,35′-dodecaoxaoctatriacontan-38′-yl)fluorene(0.25 mmol), 1,4-divinylbenzene (32.3 mg, 0.25 mmol), palladium acetate(3 mg, 0.013 mmol), tri-ortho-tolylphosphine, (10 mg, 0.033 mmol), andpotassium carbonate (162 mg, 1.2 mmol) are combined with 5 mL of DMF ina small round bottom flask charged with a Teflon coated stirbar. Theflask is fitted with a needle valve and put in a Schlenk line. Thesolution is degassed by three cycles of freezing, pumping, and thawing.The mixture is then heated to 100° C. overnight. The polymer can besubsequently reacted with terminal linkers or capping units usingsimilar (in situ) protocols to those provided in the previous examples(9, 10 and 11) or by modifying them post polymerization work up as aseparate set of reactions.

Example 16: Synthesis of a Branched Phenylene Vinylene Co-Polymer

2,7-dibromo-9,9-bis(3,5-(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yl)benzyl)-9H-fluorene (636 mg, 0.25 mmol), 1,4-divinylbenzene (32.3 mg,0.25 mmol), palladium acetate (3 mg, 0.013 mmol),tri-ortho-tolylphosphine, (10 mg, 0.033 mmol), and potassium carbonate(162 mg, 1.2 mmol) were combined with 5 mL of DMF in a small roundbottom flask charged with a Teflon coated stirbar. The flask was fittedwith a needle valve and put in a Schlenk line. The solution was degassedby three cycles of freezing, pumping, and thawing. The mixture was thenheated to 100° C. overnight. The polymer can be subsequently reactedwith terminal linkers or capping units using similar (in situ) protocolsto those provided in Example 5 or by modifying them post polymerizationwork up as a separate set of reactions.

Example 17: Synthesis of a Branched Phenylene Vinylene Co-Polymer withFunctional Amines for Covalent Conjugation. Poly[2,7-divinyl{9,9-bis(3,5-(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yl)benzyl)-9H-fluorene}-alt-1,4-benzene-co-4-phenoxybutyl-N-t-butylcarbamate]

Step 1: Polymerization

2,7-dibromo-9,9-bis(3,5-(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yl)benzyl)-9H-fluorene (636 mg, 0.25 mmol), 1,4-divinylbenzene (32.3 mg,0.25 mmol), palladium acetate (3 mg, 0.013 mmol),tri-ortho-tolylphosphine, (10 mg, 0.033 mmol), and potassium carbonate(162 mg, 1.2 mmol) were combined with 5 mL of DMF in a small roundbottom flask charged with a Teflon coated stirbar. The flask was fittedwith a needle valve and put in a Schlenk line. The solution was degassedby three cycles of freezing, pumping, and thawing. The mixture was thenheated to 100° C. overnight.

Step 2: Linker Addition

The next morning divinylbenzene (10 mg, 0.077 mmol) was transferred to asmall round bottom flask with 1 mL of DMF. The flask was fitted with aneedle valve and put in a Schlenk line. The solution was degassed bythree cycles of freezing, pumping, and thawing. The solution wastransferred via cannula through the needle valves and into thepolymerization reaction. After this addition the reaction was allowed tocontinue at 100° C. overnight. The next day tert-butyl4-(4-bromophenoxy)butylcarbamate (53 mg, 0.15 mmol) and 1 mL of DMF weretransferred to a small round bottom flask. The flask was fitted with aneedle valve and put in a Schlenk line. The solution was degassed bythree cycles of freezing, pumping, and thawing. The solution wastransferred via cannula through the needle valves and into thepolymerization reaction. After this addition the reaction was allowed tocontinue at 100° C. overnight.

Step 3: Work Up

The reaction is then cooled and diluted with 100 mL of water. Theaqueous solution was filtered twice through G-6 glass fiber filterpaper. The filtrate was evaporated to dryness and re-diluted withdichloromethane. The organic layer was dried over MgSO4 and filtered.The filtrate was evaporated to yield an amber colored oil (342 mg, 56%yield).

A 4 mL portion of 4M HCl in dioxane was added to the polymer residue andstirred for a minimum of 4 hours. The solution was neutralized with 2 Mpotassium carbonate (aq) and then the solvent was removed under vacuum.The resulting residue was diluted to ˜30 mL with 20% ethanol in waterand filtered. Preparative gel permeation chromatography is performedwith G-25 desalting medium to remove excess salts from the polymer.Solvent in the fractions is removed with rotary evaporation and thepolymer is collected as an amber oil.

The linker or capping unit addition steps can be performed in thepolymerization reaction as presented above or alternatively, in someembodiments, can be performed in a separate set of reactions after thepolymerization work up. In the latter case, the polymer is reacted underthe analogous conditions as those provided in the example. In otherembodiments, it is also possible to react with a combination of terminalmonomers to introduce polymers with bi-functionality, allowing thepolymer to be conjugated to more than one entity.

Example 18: Synthesis of a Fluorene Monomer with Glycerol-BasedDendrimers

Step 1

Dimethyl 3,3′-(2,7-dibromo-9H-fluorene-9,9-diyl)dipropanoate.2,7-Dibromofluorene (1 g, 3.1 mmol), methyl acrylate (861 mg, 10 mmol)tetrabutylammonium bromide (100 mg, 0.3 mmol) and toluene (5 mL) wereadded to a small round bottom flask with a Teflon-coated stirbar. Next 2mL of 50% NaOH (aq) is added while stirring. The reaction is allowed toproceed overnight. The next day the toluene layer is transferred to aflask and the aqueous layer extracted with two portions of toluene. Theorganic layers are combined, dried with Mg2SO4, and filtered. Silica (2g) is added to the filtrate and the solution is evaporated. The productis obtained as a white solid (1.23 g, 80% yield) after purification bycolumn chromatography.

Step 2

3,3′-(2,7-dibromo-9H-fluorene-9,9-diyl)dipropanoic acid. Dimethyl3,3′-(2,7-dibromo-9H-fluorene-9,9-diyl)dipropanoate (1.23 g, 2.5 mmol)is transferred to a small round bottom flask equipped with aTeflon-coated stirbar. A mixture of THF:MeOH:H2O, 3:2:1, (10 mL) isadded and the mixture is stirred for 1 hr. Then a 1 mL portion of 1MNaOH (aq) is added and the mixture is stirred overnight. The next daythe water layer is isolated and extracted with 20 mL portions diethylether three times. Next the water layer is acidified to ˜pH 2. The waterlayer is extracted three times with 20 mL portions of dichloromethane.The organic layers are combined and dried with Mg2SO4. The organicsolution is filtered and the solvent evaporated to obtain the product asan off-white solid (948 mg, 90% yield).

Step 3

3,3′-(2,7-Dibromo-9H-fluorene-9,9-diyl)bis(N-(7,15-bis((2,3-dihydroxypropoxy)methyl)-1,3,19,21-tetrahydroxy-5,9,13,17-tetraoxahenicosan-11-yl)propanamide).3,3′-(2,7-Dibromo-9H-fluorene-9,9-diyl)dipropanoic acid (500 mg, 1.1mmol),11-amino-7,15-bis((2,3-dihydroxypropoxy)methyl)-5,9,13,17-tetraoxahenicosane-1,3,19,21-tetraol(1.954, 3.3 mmol) (prepared as per ref. Heek, T.; Fasting, C.; Rest, C.;Zhang, X.; Wurthner, F.; Haag, R. Chem. Commun., 2010, 46, 1884-1886),and N,N′-dimethylaminopyridine (61 mg, 0.5 mmol) are combined in a roundbottom flask equipped with a Teflon-coated stirbar and sealed with arubber septum. The flask was flushed with N2 and 10 mL of anhydrousdichloromethane was added via syringe. The mixture is stirred todissolve the solids. In another round bottom flask equipped with aTeflon-coated stirbar, dicyclohexylcarbodiimide (DCC, 910 mg 4.4 mmol)transferred and the flask is sealed with a rubber septum. Next, 5 mL ofanhydrous dichloromethane is transferred to the flask via syringe. TheDCC solution is transferred to the fluorene reaction mixture via asyringe dropwise. The reaction is allowed to react overnight. The nextday the reaction mixture is filtered. The filtrate is purified by columnchromatography to afford a clear oil (1.24 g, 70% yield).

Example 19: Synthesis of a Fluorene Monomer PAMAM-Based Dendritic SideChain Capped with methylPEG Chains

Step 1: 9,9′-(3,3′-Diamido(tetramethyl PAMAM G[2])-2,7-dibromofluorene(i)

Dimethyl 3,3′-(2,7-dibromo-9H-fluorene-9,9-diyl)dipropanoate (g, 2.0mmol) is transferred to a round bottom flask equipped with a stirbar andsealed with a rubber septum. The flask is flushed with nitrogen and 10mL of dry methanol is transferred to the flask via syringe and the solidis dissolved by stirring. Ethylenediamine (5.5 mL, 82 mmol) is added viasyringe slowly and the mixture is allowed to stir for 2 hours. Theseptum is removed and the methanol and unreacted ethylenediamine isremoved under vacuum. Another 10 mL portion of methanol is added andstirred and then was evaporated to remove any remaining ethylenediamine.The residue remaining in the flask was then sealed again with a septum,flushed with nitrogen, and dry methanol (10 mL) was added and stirred.Methyl acrylate (7.2 mL, 80 mmol) is added slowly via syringe and themixture is allowed to stir for 2 hours. The septum is again removed andthe methanol and methyl acrylate are removed under vacuum. A 10 mLportion of toluene is added, the mixture stirred, and the solventremoved under vacuum affording an off-white solid (1.79 g, quantitativeyield).

Step 2: 9,9′-(3,3′-Diamido(PAMAM G[2] tetraacid)-2,7-dibromofluorene(ii)

9,9′-(3,3′-Diamido(tetramethyl PAMAM G[2])-2,7-dibromofluorene (i) (1.79g, 2 mmol) is transferred to a small round bottom flask equipped with aTeflon-coated stirbar. A mixture of THF:MeOH: H₂O, 3:2:1, (10 mL) isadded and the mixture is stirred for 1 hr. Then a 1 mL portion of 1MNaOH (aq) is added and the mixture is stirred overnight. The next daythe water layer is isolated and extracted with 20 mL portions diethylether three times. Next the water layer is acidified to ˜pH 2. The waterlayer is extracted three times with 20 mL portions of dichloromethane.The organic layers are combined and dried with Mg₂SO₄. The organicsolution is filtered and the solvent evaporated to obtain the product asan off-white solid (1.51 g, 90% yield).

Step 3: 9,9′-(3,3′-Diamido(PAMAM G[2]N-(2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl)propionamidyl)-2,7-dibromofluorene(iii)

9,9′-(3,3′-Diamido(PAMAM G[2] tetraacid)-2,7-dibromofluorene (ii) (500mg, 0.6 mmol), 2,5,8,11,14,17,20,23-octaoxapentacosan-25-amine (1.15 g,3 mmol)), and N,N′-dimethylaminopyridine (12 mg, 0.1 mmol) are combinedin a round bottom flask equipped with a Teflon-coated stirbar and sealedwith a rubber septum. The flask was flushed with N₂ and 10 mL ofanhydrous dichloromethane was added via syringe. The mixture is stirredto dissolve the solids. In another round bottom flask equipped with aTeflon-coated stirbar, dicyclohexylcarbodiimide (DCC, 825 mg 4.0 mmol)transferred and the flask is sealed with a rubber septum. Next, 5 mL ofanhydrous dichloromethane is transferred to the flask via syringe. TheDCC solution is transferred to the fluorene reaction mixture via asyringe dropwise. The reaction is allowed to react overnight. The nextday the reaction mixture is filtered. The filtrate is purified by columnchromatography to afford a clear oil (967 g, 70% yield).

Example 20: Synthesis of a Fluorene Monomer with Highly BranchedPEGylated Side Chains Based on a Trihydroxybenzene Linkage

Step 1: Methyl3,4,5-tris(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yloxy)benzoate(iv)

Methyl 3,4,5-trihydroxybenzoate (200 mg, 1.1 mmol),2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yl4-methylbenzenesulfonate (2.58 g, 3.85 mmol), and 18-crown-6 (100 mg,0.38 mmol) are transferred to a round bottom flask equipped with aTeflon-coated stirbar. Acetone (10 mL) is added and the flask isequipped with a reflux condenser. The mixture is refluxed with constantstirring overnight. The next day silica (4 g) is added and the solventevaporated. After purification by column chromatography, a clear oil isobtained (887 mg, 48% yield).

Step 2:3,4,5-Tris(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yloxy)-N-(2-aminoethyl)benzamide(v)

Methyl3,4,5-tris(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yloxy)benzoate(iv) (887 mg, 0.52 mmol) flask is transferred to a round bottom flaskequipped with a stirbar and sealed with a rubber septum. The flask isflushed with nitrogen and 10 mL of dry methanol is transferred to theflask via syringe and the solid is dissolved by stirring.Ethylenediamine (0.7 mL, 10.4 mmol) is added via syringe slowly and themixture is allowed to stir for 2 hours. The septum is removed and themethanol and unreacted ethylenediamine is removed under vacuum. Theproduct is obtained as an oil (886 mg, quanitative yield).

Step 3:]3,3′-(2,7-Dibromo-9H-fluorene-9,9-diyl)bis(N-(2-3,4,5-tris(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yloxy)-benzamidyl-Namidoethyl)propanamide) (vi)

3,4,5-Tris(2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yloxy)-N-(2-aminoethyl)benzamide(v) (886 mg, 0.52 mmol),3,3′-(2,7-Dibromo-9H-fluorene-9,9-diyl)dipropanoic acid (112 mg, 0.24mmol), and N,N′-dimethylaminopyridine (12 mg, 0.1 mmol) are combined ina round bottom flask equipped with a Teflon-coated stirbar and sealedwith a rubber septum. The flask was flushed with N₂ and 10 mL ofanhydrous dichloromethane was added via syringe. The mixture is stirredto dissolve the solids. In another round bottom flask equipped with aTeflon-coated stirbar, dicyclohexylcarbodiimide (DCC, 148 mg 0.72 mmol)transferred and the flask is sealed with a rubber septum. Next, 5 mL ofanhydrous dichloromethane is transferred to the flask via syringe. TheDCC solution is transferred to the fluorene reaction mixture via asyringe dropwise. The reaction is allowed to react overnight. The nextday the reaction mixture is filtered. The filtrate is purified by columnchromatography to afford a clear oil (924 mg, 70% yield).

Example 21. Dual End Capped Polymer Used to Create a Polymer-Dye Labelfor Biomolecule or Substrate Conjugation

Step 1: Synthesis of an Asymmetric Neutral Water-Soluble Polymer with at-BOC Protected Amine Pendant Group at One Terminus of the Polymer

2-bromo-7-(4″-phenoxybutyl-1-tert-butyl carbamate)-poly-2,7{9,9-bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane)fluorene.

2-bromo-9,9-di(2′,5′,8′,11′,14′,17′,20′,23′,26′,29′,32′,35′-dodecaoxaoctatriacontan-38′-yl)-7-(4″,4″,5″,5″-tetramethyl-1″,3″,2″-dioxaborolan-2-yl)fluorene(1.0 g, 0.674 mmol), 3 mL of tetrahydrofuran, and 2 mL of 2M potassiumcarbonate (aqueous) were transferred to a small round bottom flaskcharged with a Teflon stirbar. The flask was fitted with a septum andthe solution is degassed by sparging with Ar for 15 minutes. Palladiumtetra(triphenylphoshine) (15.6 mg, 0.013 mmol) was added through theneck of the flask and the flask was transferred to a reflux condenserequipped with a needle valve and fixed to a Schlenk line. The solutionwas quickly frozen solid with liquid nitrogen and was further degassedusing freeze-pump-thaw technique. Once degassed the reaction was heatedto 80° C. with constant stirring. The reaction was allowed to proceedovernight. The next day tert-butyl 4-(4-bromophenoxy)butylcarbamate (35mg, 0.10 mmol) in 1 mL of THF was degassed with three freeze-pump-thawcycles and then added to the polymerization reaction via cannula underexcess nitrogen pressure. The reaction continued overnight at 80° C. Thenext day the reaction mixture was cooled and the bulk of the solvent wasremoved under vacuum. The remaining material was transferred to a smallErlenmeyer flask with a total of ˜50 mL of dichloromethane. The solutionwas stirred for 30 minutes. Approximately 1 g of MgSO₄(anhydrous) wasadded to the solution and the mixture was filtered through a flutedpaper filter. The filtrate was evaporated and 410 mg (47% yield) of anamber oil was collected.

Step 2: Synthesis to Append a Terminal Linking Monomer with a t-ButylEster at the Terminus Opposite the Protected Amine Pendant

2-(4-(tert-butyl1-phenoxy-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-oate))-7-(4″-phenoxybutyl-1-tert-butylcarbamate)-poly-2,7{9,9-bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane)fluorene.

2-Bromo-7-(4″-phenoxybutyl-1-tert-butyl carbamate)-poly-2,7{9,9-bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane)fluorene(410 mg, 0.32 mmol of repeat unit), tert-butyl1-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)-3,6,9,12,15,18,21-heptaoxatetracosan-24-oate(33 mg, 0.048 mmol), 2 mL of tetrahydrofuran, and 1.5 mL of 2M potassiumcarbonate (aqueous) were transferred to a small round bottom flaskcharged with a Teflon stirbar. The flask was fitted with a septum andthe solution is degassed by sparging with Ar for 15 minutes. Palladiumtetra(triphenylphoshine) (15 mg, 0.013 mmol) was added through the neckof the flask and the flask was transferred to a reflux condenserequipped with a needle valve and fixed to a Schlenk line. The solutionwas quickly frozen solid with liquid nitrogen and was further degassedusing freeze-pump-thaw technique. Once degassed the reaction was heatedto 80° C. with constant stirring. The reaction was allowed to proceedovernight. The remaining material was transferred to a small Erlenmeyerflask with a total of ˜50 mL of dichloromethane. The solution wasstirred for 30 minutes. Approximately 1 g of MgSO₄(anhydrous) was addedto the solution and the mixture was filtered through a fluted paperfilter. The filtrate was evaporated and 351 mg (78% yield) of an amberoil was collected.

Step 3: Synthesis of a Neutral Water-Soluble Polymer with Primary Amineat One Terminus and a t-Butyl Ester Pendant on the Other

2-(4-(tert-butyl1-phenoxy-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-oate))-7-(4″-phenoxybutyl-1-amino)-poly-2,7{9,9-bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane)fluorene.

2-(4-(tert-butyl1-phenoxy-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-oate))-7-(4″-phenoxybutyl-1-tert-butylcarbamate)-poly-2,7{9,9-bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane)fluorene(23 mg, 0.018 mmol) and 0.5 mL of 4M HCl in dioxane were combined in a 1dram vial with a Teflon-coated stirbar. The mixture was stirred for 4hours. The mixture was neutralized with 2M potassium carbonate(aqueous). The solution was then diluted to 50 mL of roughly 20% ethanolin water and filtered through G-6glass fiber filter paper. The filtratewas desalted by centrifugation in a 4 mL 10 KDa cutoff centrifugefilter. The retentate was evaporated under vacuum and two 1 mL portionsof toluene were added and removed under vacuum to remove any remainingwater. A thick amber liquid was recovered from the desalting (21 mg, 85%yield).

Step 4: Attachment of an NHS-Functionalized Dye to a Primary AminePendant on a Neutral Water-Soluble Polymer

2-(4-(tert-butyl1-phenoxy-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-oate))-7-(4″-phenoxybutyl-1-amido-DYE)-poly-2,7{9,9-bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane)fluorene.

2-(4-(tert-butyl1-phenoxy-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-oate))-7-(4″-phenoxybutyl-1-amino)-poly-2,7{9,9-bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane)fluorene(518 ug, 0.4 μM) was dissolved in 100 μL of dry dichloromethane in aglass vial. A small crystal of 4-N,N′-dimethylaminopyridine was added.In another vial 65 μg (0.06 □uM) of NHS-functionalized DyLight 594(Pierce) was dissolved in 50 μL of dry dichloromethane. The twosolutions were combined and allowed to stir in a sealed vial for 4 hourscovered in foil. The solvent was then evaporated and the remainingmaterial was dissolved in 95% ethanol and injected onto a Sepharose 6column. The remaining dye was separated from the polymer. A solution ofdye-labeled polymer was obtained from combining fractions (˜100 μg, 20%yield).

Step 5: Hydrolysis of the t-Butyl Ester Pendant on the Dye-LabeledNeutral Water-Soluble Polymer to Form the Carboxylic Acid Pendant on Oneof the Termini

2-(4-(1-phenoxy-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-acid))-7-(4″-phenoxybutyl-1-amido-DYE)-poly-2,7{9,9-bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane)fluorene.

The polymer was combined with ZnBr₂ in dichloromethane and stirredovernight. The next day a portion of water was added and the mixture wasstirred for 1 hour. The solvent was evaporated and the residue wasdissolved in 20% ethanol in water. The filtrate was then desalted bycentrifugation in a 4 mL 10 KDa cutoff centrifuge filter. The retentatewas evaporated under vacuum and two 1 mL portions of toluene were addedand removed under vacuum to remove any remaining water.

Activation (for subsequent conjugation) of the second functional groupin this example (carboxylic acid) can be achieved using a number ofdifferent methods including those described in Examples 29 and otherexamples with carboxylic acid to amine to maleimide. One such method isgiven below in Step 6, by way of example only.

Step 6: NHS Activation of the Carboxylic Acid Penant of a Dye-LabeledNeutral Water-Soluble Polymer

2-(4-(1-phenoxy-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-N-hydroxysuccinimidylester))-7-(4″-phenoxybutyl-1-amido-DYE)-poly-2,7{9,9-bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane)fluorene.2-(4-(1-phenoxy-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-acid))-7-(4″-phenoxybutyl-1-amido-DYE)-poly-2,7{9,9-bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane)fluoreneand O—(N-Succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate andDIPEA are combined in dry acetonitrile and allowed to react undernitrogen for 30 min. The solution is evaporated and the solid isresuspended in dry dichloromethane. Solids are filtered off and thefiltrate is evaporated to afford the NHS ester.

In further embodiments, various commonly used protecting groups can beused with those functional groups provided (amine and carboxylic acid).Additionally different capping monomers and protecting groupcombinations can be used to produce polymers with different functionalgroups for conjugation. Eliminating or substituting the dye labelingstep for another entity will result in a polymer with two differentfunctional groups for conjugation. The dye attachment via NHS/aminechemistry can be performed under a variety of commonly used conditions.Dye attachment can also be performed with other functional chemistries.

Example 22. Asymmetric Polyfluorene Synthesis Using Non-Regulated SuzukiConditions

Step 1: Polymerization

Method A:

A solution of K₂CO₃ in water (2M, 4 mL) was added to a stirred mixtureof2-bromo-9,9-di(2′,5′,8′,11′,14′,17′,20′,23′,26′,29′,32′,35′-dodecaoxaoctatriacontan-38′-yl)-7-(4″,4″,5″,5″-tetramethyl-1″,3″,2″-dioxaborolan-2-yl)fluorene(A) (2.3 g, 1.5 mmol) and THF (6 mL) in a round bottom flask. Thismixture was degassed with argon for 15 min. Palladiumtetrakis(triphenylphosphine) (38.5 mg, 0.03 mmol) was added to themixture and the flask was attached to a reflux condenser. The reactionvessel was degassed via 3 freeze-pump-thaw cycles and then heated to 80°C. for 12 h.

The reaction mixture was cooled to 23° C. and solvent removed by rotaryevaporation. The resulting residue was transferred to an Erlenmeyerflask and diluted with 20% EtOH/H2O (75 mL). EDTA (300 mg, 1.0 mmol) wasadded to the mixture and stirred at 23° C. for 1 h. The mixture wasfiltered through a glass fiber filter paper and the filter paper rinsedwith 20% EtOH/H₂O. The resulting filtrate was then filtered through a0.45 um cup filter.

The filtered reaction mixture was purified using tangential flowfiltration (TFF) and was diafiltered into 20% ethanol using a 10,000molecular weight cutoff membrane (regenerated cellulose Prep/Scale TFFcartridge system, Millipore, Billerica, Mass.) until conductivity of thefiltrate measured less than 0.01 mS/cm. The solvent was then removedunder vacuum to give poly[2,7{9,9-bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane)fluorene](B) as a gel-like product (1.41 g, 71%) Molecular weight determined byGPC analysis relative to polystyrene standards (Mn=51,000, Mw=108,000,Mp=90,000, D=2.1). The extent of end linker incorporation was determinedby first converting the acid to an NHS ester (similar protocol to thatprovided in Example 29) then reacting with an amine functional dye.After purification of free dye the ratio of dye to polymer wasdetermined from absorbance measurements, factoring in the difference inextinction coefficients and polymer molecular weight.

Method B:

A solution of K₂CO₃ in water (2M, 4 mL) was added to a stirred mixtureof2-bromo-9,9-di(2′,5′,8′,11′,14′,17′,20′,23′,26′,29′,32′,35′-dodecaoxaoctatriacontan-38′-yl)-7-(4″,4″,5″,5″-tetramethyl-1″,3″,2″-dioxaborolan-2-yl)fluorene(A) (2.3 g, 1.5 mmol) and DMF (6 mL) in a round bottom flask. Thismixture was degassed with argon for 15 min. Palladiumtetrakis(triphenylphosphine) (38.5 mg, 0.03 mmol) was added to themixture and the flask was attached to a reflux condenser. The reactionvessel was degassed via 3 freeze-pump-thaw cycles and then heated to 80°C. for 12 h. Work-up and purification was performed in a manner similarto previously described Method A. Molecular weight determined by GPCanalysis relative to polystyrene standards (Mn=96,000, Mw=231,000,Mp=185,000, D=2.4).

Method C:

Cs₂CO₃ (2.08 g, 6.4 mmol) was added to a stirred mixture of2-bromo-9,9-di(2′,5′,8′,11′,14′,17′,20′,23′,26′,29′,32′,35′-dodecaoxaoctatriacontan-38′-yl)-7-(4″,4″,5″,5″-tetramethyl-1″,3″,2″-dioxaborolan-2-yl)fluorene(A) (200 mg, 0.135 mmol) and DMF (7 mL) in a round bottom flask. Thismixture was degassed with argon for 15 min. Palladiumtetrakis(triphenylphosphine) (15.6 mg, 10 mol %) was added to themixture and the flask was attached to a reflux condenser. The reactionvessel was degassed via 3 freeze-pump-thaw cycles and then heated to 80°C. for 12 h. Work-up and purification was performed in a manner similarto previously described Method A. Molecular weight determined by GPCanalysis relative to polystyrene standards (Mn=95,000, Mw=218,000,Mp=206,000, D=2.3).

Step 2: End Capping

-(4-iodophenyl)butanoic acid (227 mg, 0.783 mmol) was washed into aflask containing poly[2,7{9,9-bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane)fluorene](B)(1.00 g, 0.783 mmol) using THF (3.5 mL). A solution of K₂CO₃ in water(2M, 2.3 mL) was added to the flask and this mixture was degassed withargon for 15 min. Palladium tetrakis(triphenylphosphine) (36 mg, 4 mol%) was added to the mixture and the flask was attached to a refluxcondenser. The reaction vessel was degassed via 3 freeze-pump-thawcycles and then heated to 80° C. for 12 h.

The reaction mixture was cooled to 23° C. and the solvent removed withrotary evaoporation. The resulting residue was transferred to anErlenmeyer flask and diluted with 20% EtOH/H2O (150 mL). EDTA (500 mg)was added to the mixture and stirred at 23° C. for 1 h. The mixture wasfiltered through a glass fiber filter paper and the filter paper rinsedwith 20% EtOH/H₂O. The resulting filtrate was then filtered through a0.45 um cup filter.

The filtered reaction mixture was purified using tangential flowfiltration (TFF) and was diafiltered into 20% ethanol using a 10,000molecular weight cutoff membrane (regenerated cellulose Prep/Scale TFFcartridge system, Millipore, Billerica, Mass.) until conductivity of thefiltrate measured less than 0.01 mS/cm. The solvent was then removedunder vacuum to give 4-(Poly[2,7{9,9-bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane)fluorene]yl)phenyl)butanoicacid (C) as a gel-like product (388 mg, 39%) Molecular weight determinedby GPC analysis relative to polystyrene standards (Mn=89,000,Mw=196,000, Mp=124,000, D=2.2). The extent of end linker incorporationwas determined by first converting the acid to an NHS ester (similarprotocol to that provided in Example 29) then reacting with an aminefunctional dye. After purification of free dye the ratio of dye topolymer was determined from absorbance measurements, factoring in thedifference in extinction coefficients and polymer molecular weight.

Step 3: Amine Activation

4-(Poly[2,7{9,9-bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane)fluorene]yl)phenyl)butanoicacid (C) (200 mg, 0.156 mmol) was dissolved in 2 mL ethanol, then addeddrop-wise to 23 mL of MES buffer (50 mM, pH 5) at 4° C. while stirring.N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (576 mg,3.00 mmol) was added in portions, followed by N-hydroxy succinimide (115mg, 1.00 mmol) in one portion. The solution was stirred for 30 minutes,ethylene diamine (0.501 mL, 7.50 mmol) was added drop-wise and thereaction mixture was stirred overnight at room temperature. The reactionmixture was then desalted over a G25 desalting column and the solventremoved via rotary evaporation to give N-(2-aminoethyl)-4-(Poly[2,7{9,9-bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane)fluorene]yl)butanamideas a clear yellow oil (190 mg, 95%). Molecular weight determined by GPCanalysis relative to polystyrene standards (Mn=89,000, Mw=196,000,Mp=124,000, D=2.2). Extent of amine conversion was determined byreacting the amine polymer with an NHS active dye in similar fashion asthat described in Example 38.

Example 23. Asymmetric Polyfluorene Synthesis Using Linker Modified EndCaps to Regulate the Suzuki Polymerization

Step 1: Polymerization/End Capping/Work-Up

A solution of K₂CO₃ in water (2M, 4 mL) was added to a stirred mixtureof2-bromo-9,9-di(2′,5′,8′,11′,14′,17′,20′,23′,26′,29′,32′,35′-dodecaoxaoctatriacontan-38′-yl)-7-(4″,4″,5″,5″-tetramethyl-1″,3″,2″-dioxaborolan-2-yl)fluorene(A) (2.3 g, 1.55 mmol),4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)butanoic acid(B) (6.7 mg, 2 mol %), and DMF (6 mL) in a round bottom flask equippedwith a side-arm stopcock. This mixture was degassed with argon for 25min. Palladium tetrakis(triphenylphosphine) (38.5 mg, 2 mol %) was thenadded to the mixture and the flask was attached to a reflux condenser.The reaction vessel was further degassed via 3 freeze-pump-thaw cyclesand then heated to 80° C.

Separately,4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)butanoic acid(B) (230 mg, 0.793 mmol) was dissolved in DMF (3 mL) in a found bottomflask equipped with a side arm stopcock. This solution was sparged withargon for 15 minutes, attached to a reflux condenser, and degassed viathree freeze-pump thaw cycles. Upon thawing the solution was added tothe reaction mixture after two hours of reaction time using an argonflushed syringe. The reaction mixture was stirred for an additional 12 hat 80° C.

The reaction mixture was cooled to 23° C. and solvent removed withrotary evaporation. The resulting residue was transferred to anErlenmeyer flask and diluted with 20% EtOH/H₂O (75 mL). EDTA (300 mg,1.00 mmol) was added to the mixture and stirred at 23° C. for 1 h. Themixture was filtered through a glass fiber filter paper and the filterpaper rinsed with 20% EtOH/H₂O. The resulting filtrate was then filteredthrough a 0.45 um cup filter.

The filtered reaction mixture was purified and size fractionated usingtangential flow filtration (TFF) and was diafiltered into 20% ethanolusing a 30,000 molecular weight cutoff membrane (polyethersulfonePrep/Scale TFF cartridge system, Millipore, Billerica, Mass.) untilconductivity of the filtrate measured less than 0.01 mS/cm and M_(n) ofthe retentate measured more than 70,000 by GPC. The solvent was thenremoved under vacuum to give 4-(Poly[2,7{9,9-bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane)fluorene]yl)phenyl)butanoicacid as a gel-like product (1.41 g, 71%). Molecular weight determined byGPC analysis relative to polystyrene standards (Mn=68,000, Mw=134,000,Mp=122,000, D=1.9). The extent of end linker incorporation wasdetermined by first converting the acid to an NHS ester (similarprotocol to that provided in Example 29) then reacting with an aminefunctional dye. After purification of free dye the ratio of dye topolymer was determined from absorbance measurements, factoring in thedifference in extinction coefficients and polymer molecular weight.

Despite having a molecular weight in excess of 50,000 g/mole the polymeris soluble in both water and phosphate buffered saline solutions atconcentrations easily greater than 10 mg/mL. In many conjugationexperiments the polymer provided (and other described herein withsimilar structure) was concentrated to 50 mg/mL or higher which isremarkable for a neutral conjugated polymer. The moderate molecularweight also provides extinction coefficients greater than 2,500,000M⁻¹cm⁻¹. The large extinction coefficient and quantum yield of 60% (PBS)provide for exceptionally bright fluorescent reporters for use inbiological assays including their use in flow cytometry.

Step 2: Amine Activation

4-(Poly[2,7{9,9-bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane)fluorene]yl)phenyl)butanoicacid (C) (500 mg, 0.13 mmol) was dissolved in 2 mL ethanol, then addeddrop-wise to 23 mL of MES buffer (50 mM, pH 5) at 4° C. while stirring.N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride was addedin portions, followed by N-hydroxy succinimide (0.52 g) in one portion.The solution was stirred for 30 minutes, ethylene diamine (2.8 mL) wasadded drop-wise and the reaction mixture was stirred overnight at roomtemperature. The reaction mixture was then desalted over a G25 desaltingcolumn and the solvent removed via rotary evaporation to giveN-(2-aminoethyl)-4-(Poly[2,7{9,9-bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane)fluorene]yl)butanamideas a yellow oil (450 mg, 90%). Extent of amine conversion was determinedby reacting the amine polymer with an NHS active dye in similar fashionas that described in Example 38.

Example 24. Yamamoto Polymerization of PEG Modified Polyfluorene

Step 1: Yamamoto Polymerization/Work-up

In a dry box, Ni(COD)₂ (0.387 g, 1.41 mmol), 2,2′-bipyridyl (0.220 g,1.41 mmol), COD (0.152 g, 1.41 mmol) and anhydrous DMF (16 ml) wereadded to a long-neck round bottom flask. [00251]2,7-dibromo-9,9-di(2′,5′,8′,11′,14′,17′,23′,26′,29′,32′,35′-dodecaoxaoctatriacontan-38′-yl)fluorene(A) (1.00, 0.696) was weighed into a 40 ml vial and dissolved inanhydrous DMF (8 ml). The flask was sealed with a septum and the vialwas closed with a septum screw cap. The catalyst mixture and the monomersolution were transferred out of the dry box and were placed understatic argon. The reaction flask was heated to 70° C. for 45 min. Themonomer solution was then was quickly transferred from the vial to thecatalyst mixture flask with an argon flushed syringe. The reactionmixture was then heated to 70° C. for 6 h.

The reaction mixture was cooled and solvent removed by rotaryevaporation. The resultant black residue was re-dissolved in 20% EtOH(80 mL) and centrifuged at 2400 rpm for 12 hours. The supernatant wasthen decanted and filtered through a 0.45 um cup filter.

The filtered reaction mixture was purified using tangential flowfiltration (TFF) and was diafiltered into 20% ethanol using a 10,000molecular weight cutoff membrane (polyethersulfone Prep/Scale TFFcartridge system, Millipore, Billerica, Mass.) until GPC analysis ofretentate indicated the absence of low molecular weight material. Thesolvent was then removed under vacuum to give poly[2,7{9,9-bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane)fluorene](B) as a viscous oil (0.700 g, 79%) Molecular weight determined by GPCanalysis relative to polystyrene standards (Mn=62,000, Mw=127,000,Mp=93,000, D=2.0).

Step 2

End Capping: End capping is performed in a manner similar to Example 22,Step 2.

Step 3

Amine Activation: amine activation is performed in a manner similar toExample 22, Step 3.

Example 25. Synthesis of a Tandem Polymer with Two Different Linkers

Step 1: Polymerization

In a dry box, Ni(COD)₂ (0.765 g, 2.78 mmol), 2,2′-bipyridyl (0.435 g,2.78 mmol), COD (0.301 g, 2.78 mmol) and anhydrous DMF (20 ml) wereadded to a long-neck round bottom flask.2,7-dibromo-9,9-di(2′,5′,8′,11′,14′,17′,23′,26′,29′,32′,35′-dodecaoxaoctatriacontan-38′-yl)fluorene(A) (1.80, 1.26 mmol) and tert-butyl4-(2,7-dibromo-9-methyl-9H-fluoren-9-yl)butylcarbamate (B) (0.071 g,0.126 mmol) were added to a 40 ml vial and dissolved in anhydrous DMF(30 ml). The flask was sealed with a septum and the vial was closed witha septum screw cap. The catalyst mixture and the monomer solution weretransferred out of the dry box and were placed under static argon. Thereaction flask was heated to 70° C. for 45 min. The monomer solution wasthen was quickly transferred from the vial to the catalyst mixture flaskwith an argon flushed syringe. The reaction mixture was then heated to70° C. for 6 h.

The reaction mixture was cooled and solvent removed by rotaryevaporation. The resultant black residue was re-dissolved in 20% EtOH(80 mL) and centrifuged at 2400 rpm for 12 hours. The supernatant wasthen decanted and filtered through a 0.45 um cup filter.

The filtered reaction mixture was purified using tangential flowfiltration (TFF) and was diafiltered into 20% ethanol using a 10,000molecular weight cutoff membrane (polyethersulfone Prep/Scale TFFcartridge system, Millipore, Billerica, Mass.) until GPC analysis ofretentate indicated the absence of low molecular weight material. Thesolvent was then removed under vacuum to give2,7-dibromo-poly-[2,7{9,9-bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane)fluorene-co-2,7-(9-methyl-9′-(butyl-4-t-butylcarbamate)fluorene)](C) as a viscous oil (1.3 g, 45%) Molecular weight determined by GPCanalysis relative to polystyrene standards (Mn=72,000, Mw=156,000,Mp=138,000, D=2.1).

Step 2: End Capping

A solution of K₂CO₃ in water (2M, 4 mL) was added to a stirred mixtureof 2,7-dibromo-poly-[2,7{9,9-bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane)fluorene-co-2,7-(9-methyl-9′-(butyl-4-t-butylcarbamate)fluorene)](C) (800 mg, 0.67 mmol),4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)butanoic acid(D) (120 mg, 0.41 mmol), and DMF (6 mL) in a round bottom flask. Thismixture was degassed with argon for 15 min. Palladiumtetrakis(triphenylphosphine) (50 mg, 6 mol %) was added to the mixtureand the flask was attached to a reflux condenser. The reaction vesselwas degassed via 3 freeze-pump-thaw cycles and then heated to 80° C. for12 h.

The reaction mixture was cooled to 23° C. and concentrated in vacuo to avolume of 2 mL. The crude reaction mixture was transferred to anErlenmeyer flask and diluted with 20% EtOH/H2O (75 mL). EDTA (300 mg,2.00 mmol) was added to the mixture and stirred at 23° C. for 1 h. Themixture was filtered through a glass fiber filter paper and the filterpaper rinsed with 20% EtOH/H₂O. The resulting filtrate was then filteredthrough a 0.45 um cup filter.

The filtered reaction mixture was purified and size fractionated usingtangential flow filtration (TFF) and was diafiltered into 20% ethanolusing a 10,000 molecular weight cutoff membrane (polyethersulfonePrep/Scale TFF cartridge system, Millipore, Billerica, Mass.) untilconductivity of the filtrate measured less than 0.01 mS/cm. The solventwas then removed under vacuum to give4-(4-(2-bromo-poly-[2,7{9,9-bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane)fluorene-co-2,7-(9-methyl-9′-(butyl-4-t-butylcarbamate)fluorene)])phenyl)butanoicacid (E) as a yellow oil (660 mg, 82%).

Step 3: Linker Deprotection

Trifluoroacetic acid (4 mL) was added dropwise to a stirred solution of4-(4-(2-bromo-poly-[2,7{9,9-bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane)fluorene-co-2,7-(9-methyl-9′-(butyl-4-t-butylcarbamate)fluorene)])phenyl)butanoicacid (E) (200 mg, 0.169 mmol) and dichloromethane (16 mL) in a roundbottom flask. The reaction mixture was stirred at room temperature for 2hours and then concentrated in vacuo. The residue was redissolved inminimal 20% EtOH and 1M HCl was added to the solution until pH=7. Theneutralized solution was then desalted over G25 gel and the resultantmaterial was concentrated to dryness to yield a clear pale yellow oil(F).

Examples of dye incorporation, linker activation and bioconjugation arecontained in further Example 38 and related examples.

Example 26: Synthesis of a Tandem Polymer with Two Different LinkersUsing End Capping Units to Regulate the Polymerization Reaction

Step 1: Yamamoto Polymerization

In a dry box, Ni(COD)₂ (0.433 g, 8.40 mmol), 2,2′-bipyridyl (0.246 g,8.40 mmol), COD (0.170 g, 8.40 mmol) and anhydrous DMF (15 ml) wereadded to a long-neck round bottom flask.2,7-dibromo-9,9-di(2′,5′,8′,11′,14′,17′,23′,26′,29′,32′,35′-dodecaoxaoctatriacontan-38′-yl)fluorene(A) (1.00, 0.696 mmol), tert-butyl4-(2,7-dibromo-9-methyl-9H-fluoren-9-yl)butylcarbamate (B) (0.037 g,0.069 mmol), and tert-butyl 4-(4-bromophenyl)butanoate (C) (0.004 g,0.007 mmol) were added to a 40 ml vial and dissolved in anhydrous DMF(10 ml). The flask was sealed with a septum and the vial was closed witha septum screw cap. The catalyst mixture and the monomer solution weretransferred out of the dry box and were placed under static argon. Thereaction flask was heated to 70° C. for 45 min. The monomer solution wasthen was quickly transferred from the vial to the catalyst mixture flaskwith an argon flushed syringe. The reaction mixture was then heated to70° C. for 6 h.

The reaction mixture was cooled and solvent removed by rotaryevaporation. The resultant black residue was re-dissolved in 20% EtOH(80 mL) and centrifuged at 2400 rpm for 12 hours. The supernatant wasthen decanted and filtered through a 0.45 um cup filter.

The filtered reaction mixture was purified using tangential flowfiltration (TFF) and was diafiltered into 20% ethanol using a 10,000molecular weight cutoff membrane (polyethersulfone Prep/Scale TFFcartridge system, Millipore, Billerica, Mass.) until GPC analysis ofretentate indicated the absence of low molecular weight material. Thesolvent was then removed under vacuum to give tert-butyl4-(4-(2-bromo-poly-[2,7{9,9-bis(2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaoctatriacontane)fluorene-co-2,7-(9-methyl-9′-(butyl-4-t-butylcarbamate)fluorene)])phenyl)butanoate(D) as a viscous oil (664 g, 80%). Molecular weight determined by GPCanalysis relative to polystyrene standards (Mn=50,000, Mw=88,000,Mp=174,000, D=1.8).

Step 2: Linker Deprotection

Trifluoroacetic acid (6 mL) was added dropwise to a stirred solution ofPolymer (300 mg, X mmol) and dichloromethane (24 mL) in a round bottomflask. The reaction mixture was stirred at room temperature for 2 hoursand then concentrated in vacuo. The residue was redissolved in minimal20% EtOH and 1M HCl was added to the solution until pH=7. Theneutralized solution was then desalted over G25 gel and the resultantmaterial was concentrated to dryness to yield a clear pale orange oil(261 mg, 87%).

Examples of dye incorporation, linker activation and bioconjugation arecontained in further Example 38 and related examples.

Example 27. Dual Functional Asymmetric Polymer with Both Internal andTerminal Conjugation Sites Used to Create a Polymer-Dye Label forBiomolecule or Substrate Conjugation

Suzuki polymerization of2-bromo-9,9-di(2′,5′,8′,11′,14′,17′,20′,23′,26′,29′,32′,35′-dodecaoxaoctatriacontan-38′-yl)-7-(4″,4″,5″,5″-tetramethyl-1″,3″,2″-dioxaborolan-2-yl)fluoreneis performed under those conditions described in Example 23 where y % isthe mol % of the end linker used to regulate the polymerization andensure high incorporation of linker. The linker in this example is4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)butanoic acid.In this example, x mol % of the internal linker is also added to thepolymerization to incorporate the second linking site into the polymer.This method for incorporating the internal linker is generally describedin Examples 21, 25 and 26. The internal linker must be incorporatedduring the polymerization as indicated, however, it is expected that itwould be possible to add the terminal linker as a separate step asdescribed in Examples 9, 10, 11 and 21.

Example 28: Enrichment of Linker-Functionalized Polymers

The synthesis of linker-functionalized polymers can yield a mixture ofchains with and without linker functionalities. Because conjugationefficiency is expected to improve with higher purity polymers forconjugation, the methods described in this example address this byenriching for chains containing linker.

For a polymer batch containing a mixture of a COOH-modified andunmodified polymer: Dissolve polymer in 95% EtOH, then dilute with waterto a final EtOH concentration of 20%. Desalt the polymer using 10 kDaMWCO filter until conductance is <0.1 mS/cm. Inject onto Q-Sepharosecolumn, ensuring that the polymer load is suitable for the columncapacity. Pass 20% EtOH in water over column to wash out unboundpolymer. Release bound material by changing the eluting buffer to 1MNaCl in 20% EtOH in water for two column volumes to trigger the releaseof the bound polymeric material. Collect enriched material.

The polymer is passed over a strong anion exchanger such as aQ-Sepharose column. Polymer chains bearing a functional carboxylic acidgroup will bind the strong anion exchanger, and polymer that is notfunctionalized will not bind and instead will wash through. After thenon-functionalized polymer has passed through the column, the column iswashed with 1M NaCl, which triggers the release of theacid-functionalized polymer by screening the acid group from the media.By using this method, the percent functional polymer has been shown toincrease from 25% of polymer chains bearing a carboxylic acid groupto >80% of polymer chains bearing a carboxylic acid group. This increasein functional chains has been shown by analyzing the absorbance ratiosof polymer-dye conjugates pre- and post-enrichment. This procedure isalso described in Example 38. A similar process has been validated forthe enrichement of amine containing polymers. In that case an anionicexchange resin, SP Sephrose (or similar), is loaded at reducedconductivity (below 0.01 mS/cm).

Example 29: Preparation of Polymer-Streptavidin Conjugates Via NHS/AmineCoupling Example 29a: Polymer Modifications PolymerModification—Carboxylic Acid to Amine Conversion

1.35 g of a carboxylic acid terminated polymer was dissolved at in 9 mLethanol, then added dropwise to 80 mL of 4° C. 50 mM MES, pH 5 whilestirring. 0.52 g N-hydroxy succinimide was added in one portion. Oncethe N-hydroxy succinimide had dissolved, 2.3 gN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride was addedin portions. After stirring the solution for 30 minutes, 2.8 mL ofethylene diamine was added dropwise. The solution was stirred overnightat room temperature and purified by tangential flow filtration (MWCO=10kDa). 1.22 g yield (90%).

Polymer Modification—Amine to Carboxylic Acid Conversion

70 mg of an amine-terminated polymer was dissolved in 7 mL DMSO. 2.3 mgDIPEA was added to the polymer solution. 2.2 mg DMAP was dissolved in220 μL DMSO and added to the resulting polymer solution. 5.5 mg succinicanhydride was dissolved in 550 μL DMSO and added to the resultingpolymer solution. The solution was agitated at room temperatureovernight. The reaction was then purified over Amicon Ultra centrifugalfiltration units (MWCO=10 kDa) with 25 mM MES pH 5 buffer. 62 mg yield(89%).

Polymer Modification—Carboxylic Acid to NHS-Ester Conversion

60 mg of a carboxylic acid-terminated polymer was dissolved in 600 μLacetonitrile. 1.2 μg DIPEA was added to the polymer solution. 2.8 mgN,N,N′,N′-Tetramethyl-O—(N-succinimidyl)uronium was dissolved in 370 μLacetonitrile and added to the polymer solution. The solution wasagitated at room temperature for 15 minutes. After the reaction iscomplete, the solvent was evaporated under reduced pressure. 50 mg yield(83%).

Example 29b: Protein-Polymer Conjugation

Streptavidin protein is dissolved in 50 mM NaHCO₃ pH 8.2 buffer to makea 1 mg/mL solution. Crude activated polymer (10-15 eq or as required)solution from Step 2 is added to the aqueous streptavidin proteinsolution; the protein concentration is adjust with buffer to ensure thatthe volume of organic solvent added is <10% of the total volume, ifnecessary. The solution is agitated at room temperature for 3 hr and thereaction transferred to a Amicon Ultra filter (MWCO=10 kDa) to removeDMF. The protein is recovered into the initial volume with 25 mM PO₄ pH6.5 buffer.

Purification of the Protein-Polymer Conjugate

A 1 mL HiTrap SP Sepharose FF column is equilibrated with 20 mM NaCitrate pH 3 buffer. 1 mL (0.3-1 mg/mL) of Streptavidin-polymerconjugate is loaded in 25 mM NaHPO₄ pH 6.5. The sample is wash throughcolumn with 20 mM Na Citrate pH 3 buffer until a stable baseline isobtained. Multiple 1 mL aliquots of sample may subsequently be loadedand washed. The column is washed with a minimum of 10 column volumes of20 mM Na Citrate pH 3 buffer. The conjugate is eluted with 10 columnvolumes of 20 mM Na Citrate in 0.6 M NaCl pH 7.6 buffer and the columnis stripped with 10 column volumes of 20% ethanol in the elution buffer.The elution peak is concentrated with an Amicon Ultra filter (MWCO=10kDa) to reduce the volume to ˜200 μl. A 10×300 mm Superose 12 column isequilibrated with 20 mM Na Citrate in 0.6 M NaCl pH 7.6 buffer. 200 μLof concentrated Streptavidin-polymer conjugate is loaded and eluted with20 mM Na Citrate in 0.6 M NaCl pH 7.6 buffer exchanged into PBS+0.05%NaN₃ using Amicon Ultra Centrifugation filters (10 kD MWCO). Elutionsare concentrated to desired concentration for testing; at around 2 μMStreptavidin.

Characterization of a Purified Protein-Polymer Conjugate

A 4-20% acrylamide Tris-HCl Ready Gel (BioRad) is prepared and the gelis loaded with the conjugate along with free streptavidin and freepolymer in separate lanes. Gel electrophoresis is performed in 25 mMTris 192 mM, Glycine pH 8.3 and stained with Coomassie to visualize theprotein. The gel is stained for 30 minutes then destained withcommercial destain overnight. Agarose gel conditions were also used tocharacterize polymer-streptavidin conjugates, an example which is shownin FIG. 29.

In alternative embodiments, the above example can be adapted to allowfor conjugation of the polymer to biomolecules or dyes, including butnot limited to, antibodies and nucleic acids. The amine on the polymeris converted to a maleimide and a carboxylic acid (further activated toform the NHS ester) using alternative crosslinkers or modifiers. Incertain embodiments, conjugation of the same polymer to otherbiomolecules (streptavidin, antibody fragments, nucleic acids) isfacilitated using malimide-thiol chemistry (using SATA linkers toconvert free amines on the biomolecule or TCEP (or DPP) reduction of anantibody to create free thiols).

Example 30: Preparation of Polymer-Streptavidin Conjugates ViaHydrazide/Benzaldehyde Coupling Step 1: Streptavidin-4FB Modification

Streptavidin protein is reconstituted at 1.7 mg/mL and exchange intoreaction buffer, 50 mM NaHCO₃, pH 8. 15 molar equivalents ofbifunctional benzaldehyde/succinimidyl linker, S-4FB (Solulink, SanDiego, Calif.) 20 mg/mL in anhydrous DMSO is added to streptavidin,ensuring that the organic phase is less than 10% of the total volume.Reaction is mixed on shaker for 4 hours at room temperature andunreacted linker is subsequently filtered away via Amicon Ultra filter,10 kD MWCO with 50 mM MES buffer, pH 5; centrifuged at 2400 rpm and arepeated wash×3. Streptavidin protein is recovered in its initialvolume, targeting 1.7 mg/mL in conjugation buffer, 50 mM NaPO₄, pH 6.5.

Step 2: Polymer Modification

Polymer with terminal amine group (1 molar eq) is dissolved with DMF tomake a 10 mg/mL solution. 20 molar equivalents of a bifunctionalhydrazine/succinimidyl linker, SHTH (Solulink, San Diego, Calif.) at 80mg/mL in anhydrous DMSO is added to the polymer solution. 1 drop ofDIPEA is added to the reaction by a syringe and 22 g needle. Thesolution is agitated at room temperature for 4 hr and the reactiontransferred to a Amicon Ultra filter (MWCO=10 kDa) filled with 25 mM MESpH 5 buffer. The solution is then centrifuged. The filter is refilledand washed with the following wash buffers:

-   -   1×DI H2O+1 drop 1M HCl    -   1×DI H2O+1 drop 1M NaOH    -   3×50 mM MES, pH 5

Step 3: Protein-Polymer Conjugation

15 equivalents of modified polymer from Step 2 are added with desiredamount of modified protein from Step 1. Aniline is added to the reactionfor a final concentration of 10 mM and allowed to mix for 12 hours. Thereaction is purified with Amicon Ultra filter (MWCO=10 kDa) to removeDMF and recovered with 25 mM PO₄ pH 6.5 buffer.

Step 4: Purification of the Protein-Polymer Conjugate

A 1 mL HiTrap SP Sepharose FF column is equilibrated with 20 mM NaCitrate pH 3 buffer. 1 mL (0.3-1 mg/mL) of Streptavidin-polymerconjugate is loaded in 25 mM NaHPO₄ pH 6.5. The sample is wash throughcolumn with 20 mM Na Citrate pH 3 buffer until a stable baseline isobtained. Multiple 1 mL aliquots of sample may subsequently be loadedand washed. The column is washed with a minimum of 10 column volumes of20 mM Na Citrate pH 3 buffer. The conjugate is eluted with 10 columnvolumes of 20 mM Na Citrate in 0.6 M NaCl pH 7.6 buffer and the columnis stripped with 10 column volumes of 20% ethanol in the elution buffer.The elution peak is concentrated with an Amicon Ultra filter (MWCO=10kDa) to reduce the volume to ˜200 μl. A 10×300 mm Superose 12 column isequilibrated with 20 mM Na Citrate in 0.6 M NaCl pH 7.6 buffer. 200 μLof concentrated Streptavidin-polymer conjugate is loaded and eluted with20 mM Na Citrate in 0.6 M NaCl pH 7.6 buffer. Fractions are pooled andbuffer exchanged into PBS+0.05% NaN₃ using Amicon Ultra Centrifugationfilters (10 kD MWCO). Elutions are concentrated to desired concentrationfor testing; at around 2 M Streptavidin.

Step 5: Characterization of a Purified Protein-Polymer Conjugate

A 4-20% acrylamide Tris-HCl Ready Gel (BioRad) is prepared and the gelis loaded with the conjugate along with free streptavidin and freepolymer in separate lanes. Gel electrophoresis is performed in 25 mMTris 192 mM, Glycine pH 8.3 and stained with Coomassie to visualize theprotein. The gel is stained for 30 minutes then destained withcommercial destain overnight.

FIG. 14 top, depicts conjugation of streptavidin to a polymer of formula(Vb) in cartoon format. FIG. 14, bottom, is a Coomassie stain ofacrylamide gel which depicts the mobility of the conjugate is retardedrelative to the free protein indicating an increase in mass. A neutralpolymer alone shows no evidence of staining and without a formal charge,the polymer is not mobile in the electrophoritic field.

In alternative embodiments, the above example can be adapted to allowfor conjugation of the polymer to biomolecules or dyes, including butnot limited to, antibodies and nucleic acids. The amine on the polymeris converted to a maleimide and a carboxylic acid (further activated toform the NHS ester) using alternative crosslinkers or modifiers. Incertain embodiments, conjugation of the same polymer to otherbiomolecules (streptavidin, antibody fragments, nucleic acids) isfacilitated using malimide-thiol chemistry (using SATA linkers toconvert free amines on the biomolecule or TCEP reduction of an antibodyto create free thiols) and NHS-amine chemistry (reacting the NHS polymerdirectly with lysines on the protein or nucleic acid).

Example 31: Preparation of Biotin-Labeled Polymers

Amine functionalized polymer of formula (Vc) is dissolved at 10 mg/mL inanhydrous DMF and divided into two portions. NHS-biotin (0.9 mg in 90μL, 88 equivalents) (Pierce, 20217) and NHS-LC-LC-biotin (Pierce, 21343)at 10 mg/mL (1.5 mg in 150 μL, 88 equivalents) are dissolved inanhydrous DMF. The NHS-biotin and NHS-LC-LC-biotin solutions areimmediately added to the two portions of polymer solution and allowed tomix on a shaker overnight in the dark. Excess reactant is removed bywashing the solution using Amicon Ultra-4 mL 10 kD MWCO filtercartridges in a series of wash steps: First, the cartridge is firstfilled approximately halfway with water, and the reaction solution (bypipet) subsequently added directly into the water. Next, the cartridgeis filled with water until it is full. The solution is mixed bypipetting up and down. Then, the cartridge is centrifuged at 2400 rpmfor 30 minutes, or until the volume is reduced to 250 LL. The cartridgeis then refilled with water 1 drop of 1M HCl is added; the solution ismixed, and centrifuged at 2400 rpm for 30 minutes, or until the volumewas reduced to 250 μL. Next, the cartridge is refilled with water, 1drop of 1M NaOH is added; the solution is mixed, and centrifuged at 2400rpm for 30 minutes, or until the volume is reduced to 250 μL. Thecartridge is then refilled with water, mixed and centrifuged at 2400 rpmfor 30 minutes, or until the volume is reduced to 250 μL. This finalstep is repeated for a total of 5 washes.

Characterization of a Purified Biotin-Labeled Polymer

Excess biotin-labeled polymer is incubated with a Cy5-labeledstreptavidin in DPBS buffer plus 0.2% BSA and 0.05% NaN₃. A 0.8% agarosegel is is prepared and the gel is loaded with the conjugate along withfree Cy5-streptavidin and free biotinylated polymer in separate lanes.Gel electrophoresis is performed in 10 mM NaHCO₃ pH10 and and visualizedusing a Typhoon gel imager with 457 nm and 635 nm laser excitation. FIG.15 (bottom) depicts retardation of mobility of the polymer-streptavidincomplex relative to the free protein indicating an increase in mass. Thepolymer alone shows little mobility on its own due to a lack of formalcharge.

This protocol is adapted to successfully biotin-modify a range ofconjugated polymers containing both internal and terminal amine linkers.

Example 32: Functional Testing of Covalent Polymer StreptavidinConjugates by Selective Binding to Biotinylated Microspheres

Materials Required:

1×TBST: 50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween20, pH 7.5; Biotinmicrospheres (10 mg/mL in TBST); BSA (1 mg/mL); AvDN (220 μM); andPolymer-Strepavidin (SA) conjugate: (1 μM with regard to SA, provided at5 μM).

Preparation of Master Mixes:

Prepare in labelled 1.5 mL microfuge tubes:

Experimental master mix Negative control master mix 14 μl TBST 9 μl TBST6 μl BSA stock 6 μl BSA stock 5 μl bead stock 5 μl avidin stock 5 μlbead stock

Briefly vortex both tubes and allow 20-30 minutes to pre-incubate thenegative control beads with excess avidin before proceeding. A variablespeed orbital mixer at 800 RPM for incubation is suggested to keep beadsfrom settling.

Bead Hybridization:

Pipette 10 μL of each master mix into separate labelled 1.5 ml microfugetubes. Add 2 μL of polymer-SA conjugate to each. Prepare additional tubecontaining 10 μL master mix and no polymer to be used as a blank.Briefly vortex and pulse spin all tubes. Transfer to variable speedorbital mixer and incubate for 30 mins at 800 RPM.

Bead Processing/Washing:

Add 0.5 ml TBST to all samples and controls and vortex briefly.Centrifuge at 1200 g for 2 min and remove 480 μl supernatant beingdiligent not to disturb bead pellet. Add 0.5 ml TBST to all samples andcontrols and vortex briefly. Centrifuge at 1200 g for 2 min and remove500 μl supernatant being diligent not to disturb bead pellet. Repeatsteps 3 and 4. Remove as much of remaining supernatant as possible usingP200 pipette without disturbing bead pellet. Add 100 μL TBST and vortexbriefly to re-suspend beads.

Bead Measurement:

Transfer 100 μL of positive, negative and blank beads to a BLACK 96 wellplate. Excite wells at 430 nm and collect emission in the range 450-650nm using required slit widths and/or sensitivity setting to achievemeasurable signals above background. Compare emission of positive andnegative control beads.

FIG. 16 shows the polymer streptavidin conjugate was bound to abiotinylated microsphere. Excitation at 440 nm in a florometer resultedin emission from the polymer as indicated by the solid curve. The dashedcurve represents the negative control where the biotin bead was firsttreated with excess avidin to block the biotin binding sites prior totreatment with the polymer streptavidin conjugate.

Example 33: Functional Testing of Covalent Polymer StreptavidinConjugates by Selective Binding to Biotinylated Microspheres and FRET toDye Acceptors on Co-Localized Streptavidin-Dye Conjugates

Materials Required:

1×TBST: 50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween20, pH 7.5. Biotinmicrospheres (10 mg/mL in TBST). Cy3-SA (1 μM or 50 μg/mL).Polymer-Strepavidin (SA) conjugate: (1 μM with regard to SA, provided at5 μM).

Bead Preparation and Hybridization:

Prepare in labelled 1.5 mL microfuge tubes:

Blank control Cy3-SA control FRET-SA Control 16 μl TBST 14 μl TBST 14 μlTBST 4 μl bead stock 2 μl Cy3-SA stock 2 μl Cy3-SA stock 4 μl bead stock2 μl polymer-SA stock 4 μl bead stock

Briefly vortex all tubes and transfer to variable speed orbital mixerfor incubation of at least 30 mins at 800 RPM.

Bead Processing/Washing:

Add 0.5 ml TBST to all samples and controls and vortex briefly.Centrifuge at 1200 g for 2 min and remove 480 μl supernatant beingdiligent not to disturb bead pellet. Add 0.5 ml TBST to all samples andcontrols and vortex briefly. Centrifuge at 1200 g for 2 min and remove500 μl supernatant being diligent not to disturb bead pellet. Repeatsteps 3 and 4. Remove as much of remaining supernatant as possible usingP200 pipette without disturbing bead pellet. Add 100 μL TBST and vortexbriefly to re-suspend beads.

Bead Measurement:

Transfer 100 μL of all samples to a BLACK 96 well plate. Excite wells at430 nm and collect emission in the range 450-650 nm using required slitwidths and/or sensitivity setting to achieve measurable signals abovebackground. Detect and record polymer emission in the range of 480-500nm and Cy3 emission at the expected 570 nm.

FIG. 17 shows the polymer streptavidin conjugate was bound to abiotinylated microsphere. Excitation at 440 nm in a florometer resultedin energy transfer between the polymer and a Cy3 dye conjugated to adifferent streptavidin protein as indicated by the solid upper curve.The dashed curve shows beads alone and the lower solid curve directexcitation of the Cy3-streptavidin conjugate at 440 nm.

Example 34: Functional Testing of Biotin-Labeled Polymers by SelectiveBinding to Avidin Coated Microspheres

Materials Required:

1×TBST: 50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween20, pH 7. SA microspheres(10 mg/mL in TBST). Biotin (1 mM). 440 nm biotin-polymer conjugate: (46μM).

Bead Preparation and Hybridization:

Prepare in labelled 1.5 mL microfuge tubes:

Blank control Negative control Positive Control 16 μl TBST 11 μl TBST 15μl TBST 4 μl bead stock 4 μl biotin stock 4 μl bead stock 4 μl beadstock

Briefly vortex all tubes and transfer to variable speed orbital mixerfor incubation of 20-30 mins at 800 RPM to ensure biotin has blocked allSA sites on negative control beads. Add 1 uL of polymer-biotin stock toboth positive and negative control tubes. Vortex briefly and transfer tovariable speed orbital mixer and incubate for 30 mins at 800 RPM.

Bead Processing/Washing:

Add 0.5 ml TBST to all samples and controls and vortex briefly.Centrifuge at 1200 g for 2 min and remove 480 μl supernatant beingdiligent not to disturb bead pellet. Add 0.5 ml TBST to all samples andcontrols and vortex briefly. Centrifuge at 1200 g for 2 min and remove500 μl supernatant being diligent not to disturb bead pellet. Repeatsteps 3 and 4. Remove as much of remaining supernatant as possible usingP200 pipette without disturbing bead pellet. Add 100 μL TBST and vortexbriefly to re-suspend beads.

Bead Measurement:

Transfer 100 μL of all samples to a BLACK 96 well plate. Excite wells at430 nm and collect emission in the range 450-650 nm using required slitwidths and/or sensitivity setting to achieve measurable signals abovebackground. Compare emission of positive and negative control beads.

FIG. 18 shows the biotin modified polymer was bound to a streptavidinmicrosphere (top). In FIG. 18 (bottom), excitation at 440 nm in aflorometer resulted in emission from the polymer as indicated by thesolid upper curve. The lower solid curve represents the negative controlwhere the streptavidin bead was first treated with excess biotin toblock the binding sites prior to treatment with the biontinylatedpolymer. The lower solid curve represents beads alone.

Example 35: Selective Binding of Biotin-Labeled Polymer to Dye-LabeledSA Conjugates to Validate FRET Properties and Functional Activity of thePolymer Modification

Materials Required:

Biotin-Polymer Conjugate: (46 μM). Cy3-SA conjugate (1 mg/mL or 18.9μM). BLACK 96-well plate.

Forming the Biotin-Streptavidin Complex:

In a 1.5 mL microfuge tube, combine 9.4 μL of the biotin-polymerconjugate and 2.9 μL of the Cy3-SA. Vortex to mix, then incubate on ashaker (under foil) for 0.5 h. Longer incubation times are alsosuitable.

Instrument Settings:

Model experiments were conducted on a BioTek Synergy 4 in theFluorescence mode with the following settings: Emission: 400-750 nm in 5nm steps and Sensitivity level: 50.

Plate Layout:

Prepare solutions in a BLACK 96-well plate as in the below table. Takecare to add the A+B solution last, after all other materials have beenadded:

Material Well 1 Well 2 Well 3 Polymer-biotin 9.4 μL* 9.4 μL — Cy3-SA 2.9μL* — 2.9 μL Buffer 100 μL 100 μL 100 μL *Pre-incubated in the firststep, Forming the Biotin-Streptavidin Complex.

FIG. 19 shows the biotin modified polymer was bound to a dye labeledstreptavidin (Cy3 or Texas Red—top). Excitation at 440 nm in aflorometer resulted in emission from the dye acceptors at theirrespective emission wavelength (approximately 570 nm and 620 nmrespectively—bottom left) as well as some residual emission from thepolymer (approximately 520 nm). A titration was also performed tosaturate the binding of polymer to the streptavidin (bottom right). Thesolid curve indicates the emission from the Cy3 label on thestreptavidin via energy transfer from the polymer at 440 nm excitation.The dotted curve represents the negative control where the streptavidinwas first treated with excess biotin to block the binding sites prior totreatment with the biontinylated polymer.

Example 36: Polymer-Streptavidin Conjugates for Use in Flow Cytometry

Polymer bioconjugates are evaluated by Stain Index, as defined by BectonDickinson (BD) Biosciences on a flow cytometer. See, e.g., H. Maeker andJ. Trotter, BD Biosciences Application Note: “Selecting Reagents forMulticolour Flow Cytometry”, September 2009. The stain index reports ameasure of the polymer's brightness, nonspecific binding and can also berelated by the Resolution Index on a flow cytometer. Flow cytometryprovides a method through which to measure cells of a specific phenotypeor analytes of interest on specific microspheres. This can be done withdirect labeling of a primary antibody or, if signal amplification isdesired, through a secondary antibody or the avidin-biotin complexationwith avidin-polymer conjugates.

Procedure for Cell Staining

Cells of interest are taken up in sufficient quantity, at least 10⁵ pertest condition. Cells are then spun down at 250 rcf for 3 minutes,washed in DPBS+0.2% BSA and 0.05% NaN3 (staining buffer), thenresuspended in staining buffer at 1×10⁷ cells/mL.

For primary incubation, cells are incubated with a primary conjugate(reporter labeled antibody) specific to an antigen of interest, negativecells serve as a negative non-specific binding reference. A controlpopulation or an established commercial conjugate is used as a positivecontrol. Primary polymer conjugates are incubated at 4° C. with 4×10⁵cell aliquots at concentrations with volume dilutions from 10-330 nM for30 minutes. Following primary incubation, cells are rinsed with 5volumes staining buffer and spun down at 250 rcf for 3 minutes; thisrinse is repeated three times. Cells are resuspended for testing at8×10⁵ cells/mL in DPBS+0.2% BSA, 0.05% NaN₃.

For secondary antibody labeling, an unlabeled primary antibody to theantigen of interest is incubated at 0.4 ug/uL, or other titrated amount,at 4° C. with 4×10⁵ cells per test condition for 30 min. Followingprimary incubation, cells are rinsed with 5 volumes staining buffer andspun down at 250 rcf for 3 minutes; this rinse is repeated three times.Species reactive secondary polymer conjugates are incubated at 4° C.with 4×10⁵ cell aliquots at concentrations with volume dilutions from10-330 nM for 30 minutes. Following secondary incubation, cells arerinsed with 5 volumes staining buffer and spun down at 250 rcf for 3minutes; this rinse is repeated three times. Cells are resuspended fortesting at 8×10⁵ cells/mL in DPBS+0.2% BSA, 0.05% NaN₃.

For streptavidin-polymer conjugate labeling, cells are incubated with abiotinylated primary antibody to the marker of interest, as detailedabove for the secondary antibody labeling, instead of an unlabeledprimary. Following the primary washing, cells are resuspended anddivided in 4×10⁵ cell aliquots and incubated with streptavidin-polymerconjugates at 1-100 nM volume dilutions for 30 minutes at 4° C.Following secondary incubation, cells are rinsed with 5 volumes stainingbuffer and spun down at 250 rcf for 3 minutes; this rinse is repeatedthree times. Cells are resuspended for testing at 8×10⁵ cells/mL inDPBS+0.2% BSA, 0.05% NaN₃. If further signal amplification is desired,cells and be incubated with an unlabeled primary antibody and thensubsequently follow with a species reactive biotinylated secondaryantibody prior to incubation with streptavidin conjugates. Theincubation steps, washing protocol and testing protocol should follow aspreviously detailed.

These flow testing procedures have been developed specific to CD4markers on Cyto-trol cells. Cell preparation and incubation protocolsmay vary with cell type and an optimal staining, washing and handlingprotocol should be developed specific to cell type. Workingconcentration ranges of antibodies have been identified as optimal forboth CD4 (35-50% population) and CD45 (85% population) markers onCyto-trol control lymphocytes as well as on Whole Lysed Blood (forprimary antibody only). Markers which have populations significantlydifferent than these ranges may fall outside of the suggested titrationranges.

Testing was also done on a Jurkat cell line grown in culture followingsimilar protocols. In these tests a CD45 marker was used. As there areno negative cell populations a different negative control procedure wasused. In the negative control samples the primary antibody was omittedfrom the primary incubation step. This step and all subsequent stepswere performed according to the standard protocol. Again a commerciallydye-antibody or dye-strepatvidin conjugate were used as a positivecontrol.

Procedure for Flow Cytometry Analysis

Flow testing was done in test tubes, at 0.5 mL volumes on a BD LSR IIFlow Cytometer. Flow testing is performed using the voltage settingsdetermined from daily calibration of the cytometer with calibrationparticles by flow facility staff. Lymphocyte specific gating by forwardscatter vs. side scatter is performed on unstained cell samples as abackground control. Standard doublet gating is performed for bothforward scatter and side scatter area vs. width profiles. With only asingle color, no compensation is required. Data are collected for allforward and side scatter parameters and fluorescence measurements aremade using BD's standard Pacific Blue channel. Pacific Blue datautilizes excitation with the 408 nm Violet lasers and a 450/50 BPfilter. Samples are collected for 30,000 events within the stated gatingparameters.

Representative Experiments:

CD4 marking was measured on Cyto-trol cells, lyophilized humanlymphocytes for analysis of polymer performance in flow. Cyto-trol cells(Beckman Coulter) were reconstituted in the provided reconstitutionbuffer and allowed to swell for 15 minutes at room temperature. Cellswere then spun down at 250 rcf for 3 minutes, washed in DPBS+0.2% BSAand 0.05% NaN₃ (staining/testing buffer), then resuspended in stainingbuffer at 1×10⁷ cells/mL. Cell suspension was divided in two; half thecells were incubated with biotinylated anti-CD4 at 0.4 ug/uL, the otherhalf of the cells were incubated with staining buffer as a negativecontrol for 30 min. Following primary incubation, cells were rinsed with5 volumes staining buffer and spun down at 250 rcf for 3 minutes; thisrinse was repeated three times. Cells were resuspended at prior volumein staining buffer. 4×10⁵ cells were measured per test and divided outaccordingly, streptavidin-fluorophore conjugates prepared in Example 19were incubated at 100 nM with each aliquot of cells for 30 min, allowingthe avidin-biotin complex to form. Following the secondary incubation,cells were rinsed and detailed previously. Final cell suspensions weremade for testing at 8×10⁵ cells/mL.

Flow analysis was performed on a BD LSR II flow cytometer at The ScrippsResearch Institute (TSRI), San Diego, Calif. Routine calibration withRainbow fluorescent particles for aligning fluorescent channels on thecytometer was performed by staff at TSRI, all calibrated voltages wereused, per staff recommendation. All samples were excited with a 408 nmViolet laser, the polymer conjugate was measured in the AmCyan channelwith a 525/50 filter. All samples were initially referenced to unstainedcells. The polymer streptavidin conjugate from FIG. 14 showed specificsecondary labeling of the primary identified CD4 positive cells, withthe positive cells as 44% of the population. The polymer streptavidinconjugate demonstrated a positive stain index showed low non-specificbinding with reference to unstained cells and its respective negativecontrol (FIG. 20. (A)). This provides evidence that the polymer,although its peak absorbance is a 440 nm, is a viable fluorescentmaterial for use in flow cytometry with Violet laser excitation.Secondary Antibody polymer conjugate on Cyto-trol cells

Amine-functionalized 405 polymer was conjugated to goat anti-mouse IgGκ1 purified antibody by route of maleimide-thiol conjugation and TCEPpartial reduction of the antibody. The polymer and conjugation procedureare provided specifically in Example 46.

Conjugates were tested on Cyto-trol cells (Beckman Coulter), a fixed andlypholized lymphocyte cell population for control testing of specifichuman antigens. Cell staining followed secondary cell staining protocol.Cells were incubated with and without (negative control) unlabeledanti-CD4 (RPA-T4 clone, BD Biosciences) raised in mouse against thehuman antigen. After complete washing of primary antibody incubation,cells were incubated with polymer labeled goat anti-mouse conjugates forspecific labeling of primary identified CD4 positive cells. Secondarylabeling occurs by Fc recognition and binding of the mouse primaryantibody by the secondary goat IgG, raised against murine species. Apositive control was used by incubation with commercially availablePacific Blue goat anti-mouse IgG (Invitrogen) as the secondary labelingspecies.

FIG. 20 (B) depicts the specific recognition of CD4 specific cells bythe secondary fluorescent conjugates. Unstained cells show a negativecontrol and natural autoflourescence of the cells, and incubation ofpolymer conjugate on cells with no primary labeling show minimalnon-specific binding of the conjugate to unlabeled cells. Positivecontrol, Pacific Blue goat anti-mouse shows the commercially availablestandard for CD4 labeling by secondary antibody with Violet excitation.405 polymer-goat anti-mouse conjugate (red) shows positiveidentification of CD4 positive cells, a minimal shift in the negativecell population and great fluorescent signal and resolution that PacificBlue standard.

FIG. 20 (C) depicts Streptavidin polymer conjugates on Jurkat cells.Conjugates were produced with the polymer provided in Example 11 usingthe protocol defined in Example 29. The stain index for the polymerstreptavidin conjugate was over 10 fold higher than that obtained forthe commercially available Pacific Blue streptavidin control conjugate.

FIG. 20 (D) depicts a primary monoclonal antibody polymer (antiCD4,RPA-T4) conjugate evaluated on Cyto-trol cells using the protocolsdefined above. The conjugate was prepared using the polymers andprotocols defined in Example 46. Additional details on the conjugationcan also be found in Example 39.

Example 37: Preparation of Polymer Conjugated to —COOH Beads Via EDCCoupling Materials (Per 100 μL of Beads)

LodeStars —COOH functionalized magnetic beads (Varian, Inc. PL6727-0001)(100 μL of suspension at spec'd 30 mg/mL). Polymer with amine terminalends from Example 17 (125 μL at 1.6 μM in 25 mM MES pH 5, for a 10-foldexcess over theoretical bead capacity). 10 mM NaOH (2 mL). DI H2O (3mL). 25 mM cold MES, pH 5. EDC at 50 mg/mL in 25 mM cold MES, pH 5 (100μL). NHS at 50 mg/mL in 25 mM cold MES, pH 5 (100 μL). 100 mM Tris/HClpH 7.4 (1 mL). Centrifuge and black flat-bottom 96-well plate.

Antibody capacity was given at 10 ug/mg bead, giving an amine couplingcapacity of 2 nmol polymer/mL bead (at 30 mg/mL). A 10 fold-excess ofpolymer over the suggested capacity was used to target the antibodyconcentration given in Varian's protocol.

Bead Washing

Beads were washed collectively as 600 μL and then split into 6×100 μLsamples for coupling. Beads were washed 2× with 1 mL 10 mM NaOH, then 3×with 1 mL DI H2O; in between washes, the tube was centrifuged 1 min at3000 rpm to recollect the beads as a pellet, supernatant was discardedand beads were resuspended in the next wash. After the final wash, beadswere resuspended in 600 μL cold 25 mM MES, pH 5 and aliquoted into 6×100μL volumes in microcentrifuge tubes. Beads were centrifuged again 1 minat 3000 rpm and supernatant was discarded.

EDC Activation

100 μL of the EDC solution was added to each reaction set. 100 μL of theNHS solution was added to each reaction set. Beads were resuspended byvortexing and then allowed to mix on a rotator for 30 minutes. Beadswere washed 2× in cold 25 mM MES pH 5, pelleted by centrifuging for 1min at 3000 rpm and the supernatant was discarded. Beads wereresuspended in 125 μL cold 25 mM MES, pH 5.

Polymer Coupling

125 μL of polymer at 1.6 μM was added. Samples were vortexed to mixthoroughly and then reacted at RT on a mixer for 3 hours. Beads werepelleted by centrifuging for 1 min at 3000 rpm; supernatant wasdiscarded. Beads were resuspended in 1 mL 100 mM Tris/HCl to blockunreacted —COOH sites, vortexed and mixed for 1 hour.

Beads were recollected by centrifugation and resuspended in 100 μL 25 mMMES. At this point, the supernatant of several tubes were yellow incolor and had significant absorbance at 440 nm; the beads were washed 6times until absorbance was at baseline. Beads sat for an additional 2days prior to fluorescence measurement, after sitting in solution for 2days, the supernatant was again yellow in color and had measurableabsorbance. Beads were washed 3 more times with 30 minute mixes inbetween until no absorbance was measurable. At 2 days followingfluorescence measurements, the supernatant remained clear and free ofmeasurable absorbance.

Example 38: Preparation of Polymer-Dye Conjugates Example 38a:Preparation of Polymer-Dye Conjugate at Polymer Terminal

0.5 mg amine-terminated polymer was dissolved in 15 μL DMSO. The polymersolution was then exchanged into 50 mM NaHCO3/Na2CO3, pH 8 buffer andrecovered in buffer at ˜5 mg/mL as determined by UV-VIS absorbance. 50μg NHS-ester dye (DyLight 594) was dissolved at 10 mg/mL in anhydrousDMSO, which was then immediately added to 120 μg of polymer. The tubewas mixed on shaker (600-800 rpm) for 1 h and subsequently diluted to100 μL with 20% EtOH in water. The mixture was added to a 30 cm Superdex200 SEC column in 0.6M NaCl and 20% EtOH to separate polymer-dyeconjugate from unreacted dye. The addition of dye can be used toestimate the incorporation of linker on the polymer structure bymeasuring an absorbance ratio based on the relative extinctioncoefficients of the polymer and dye. Using the molecular weight of thepolymer it is possible to estimate the number of polymer chains whichcontain a linker.

In additional embodiments, polymers with a carboxylic acid side chainare modified with amine functional dyes using standard EDC conjugationprocedures or by first converting to the NHS ester using the protocolsimilar to that described in Example 29. Thiol dyes conjugated tomaleimide terminated polymers have also been demonstrated. Any range ofchemistry pairs would be expected to work in similar fashion toconjugate a polymer and dye.

Example 38b: Preparation of Polymer-Dye Conjugate at Internal Position

In a glovebox, 100 mg polymer with internal amine functionalities wasdissolved in 10 mL anhydrous DMSO in a 20 mL amber scintillation vial.0.32 mL DIPEA was added to the polymer solution. 24 mg of NHS-ester dye(Cy3) was dissolved in 2.4 mL in anhydrous DMSO and added to the polymersolution. The vial was tightly sealed, then removed from the gloveboxand stirred at room temperature for 48 hours. The reaction was thenpurified over Amicon Ultra centrifugal filtration units (MWCO=30 kDa)with 20% ethanol in water until all free dye was removed. Purity wasverified by running a 0.15 mg sample over a 30 cm Superdex 200 SECcolumn in 0.6M NaCl and 20% ethanol. 90 mg yield (90%).

The addition of dye can be used to estimate the incorporation of linkermonomers in the polymer structure by measuring an absorbance ratio basedon the relative extinction coefficients of the polymer and dye. Forpolymers described above, the ratio of linker monomers (or dyeattachments) per fluorene monomer in the final polymer are in generalagreement with the molar feed ratio of monomers used in thepolymerization reaction.

Polymers with a carboxylic acid side chain can also be modified withamine functional dyes using standard EDC conjugation procedures or byfirst converting to the NHS ester using the protocol similar to thatdescribed in Example 29.

Analogous procedures can be used to conjugate a range of dyes includingCy3, DyLight 549, DyLight 633, FAM, FITC, Alexa633, Alexa647 and severalothers. Polymers with a carboxylic acid side chain can also be modifiedwith amine functional dyes using standard EDC conjugation procedures.

FIG. 21 (A) shows the polymer structure above conjugated to (from leftto right) FITC, Cy3, DyLight 594 and DyLight633. The polymer alone isshow for reference (far left). Note in each case the amount of residualdonor (polymer) emission is minimal. The data highlight the capabilityof generating several diagnostic signals at different wavelengths formultiplex applications. In this embodiment a single light source iscapable of generating five distinct emission wavelengths.

Example 38c: Energy Transfer Evaluation for Polymer-Dye Conjugates Basedon Polymer Excitation for Use in Polymer Tandem Conjugates

FIG. 21 (B) depicts a comparison of the fluorescence of the dye(DyLight594) excited near its absorbance maximum (lower curve) andpolymer-dye conjugate excited at 405 nm (upper curve). Dye emissionaround 620 nm was over 5 fold brighter from the polymer-dye conjugate atthe same molar concentration of dye versus direct dye excitation. Suchembodiments highlight the signal amplification afforded by the disclosedpolymer donors in energy transfer processes. The picture in the upperleft corner highlights the visual color change in the emission of thecomplex based on dye conjugation. The polymer solution emits blue in theabsence of dye and red upon dye conjugation (post purification).

FIG. 21 (C) compares the fluorescent signal of the base polymer (no dye,peak emission near 420 nm) to that of the polymer-dye conjugate (peakemission near 620 nm). The DyLight594 dye quenches>98% of the polymeremission when conjugated to the polymer above (Example 38b). This is afeature of the polymer materials as any remaining donor emission couldmanifest as background signal in multiplex assay formats. The ability toconjugate the dye directly to the polymer structure and vary the numberof attachment sites provides for efficient transfer that can beregulated by chemical design.

Example 39: Flow Testing of Monoclonal Antibody (antiCD4) Conjugates onWhole Lysed Blood Samples

Polymer conjugates of primary antiCD4 antibody (RPA-T4 clone) wereproduced using 3 different conjugation routes as provided in Examples45, 46 and 49. 1) Amine modified polymer converted to a maleimidereactive group using SMCC (maleimide/NHS crosslinker) reacted with thiolgroups on the antibody introduced by reacting SATA (thiol/NHS crosslinker) with lysine (amine) groups (CJ11-2, FIG. 22). 2) Same polymermodified with SMCC (malemide) but with thiol groups introduced onantibody using TCEP to partially reduce the disulfide linkages in theantibody (CJ13-2, FIG. 22 and FIG. 20D). 3) A carboxylic acid terminatedpolymer activated with TSTU to form the NHS ester was reacted directlywith the lysine (amine) groups on the antibody (CJ04-2 FIG. 22). Allconjugates were made from the same polymer structure and batch. Thepolymer was synthesized using the protocol depicted in Example 12 withan amine end capping unit in place of the carboxylic acid capping unitshown. The NHS/amine conjugation was done with the protocol described inExample 45. The maleimide/thiol conjugation reactions were done in lineswith those protocols described in Examples 46 and 49.

FIG. 22 depicts the performance of these conjugates in flow cytometryconduced as follows. 100 μl whole human blood from a healthy volunteerwas aliqoted into FACS tubes (duplicates for each sample). Antibodyconjugates were diluted in wash buffer (PBS with 0.5% BSA and 0.1%Sodium Azide) and added to the blood at specified concentrations.Samples were vortexed vigorously then incubated for 15-30 mins in thedark at room temperature. 2 ml of 1×BD FACS Lyse solution was added toeach sample and mixed in by vigorous vortexing prior to a further 10mins incubation in the dark at room temperature. Samples werecentrifuged for 5 min at 500 g and the supernatant tipped off anddiscarded. Samples were vortexed and 3 ml of wash buffer (PBS with 0.5%BSA and 0.1% Sodium Azide) added. Centrifugation was repeated at 500 gfor a further 5 min. The resulting supernatant was tipped off anddiscarded and the remaining cell pellet vortexed. Samples were run on aBD LSRII flow cytometer acquiring all violet channels equipped with aviolet laser and 450/50 nm filter that had been set up and precalibratedagainst BD CST beads. All polymer conjugate samples (CJ04-2, CJ11-2 andCJ13-1 lines) showed minimal non specific binding compared to unstainedcells. Further, all polymer conjugates produced significantly higherpositive signals than a commercially available Pacific Blue controlconjugate of the same antibody clone which is commonly used for flowcytometry at compatible wavelengths. The best performing conjugates fromthis set provided over 6 fold high stain index than the commerciallyavailable Pacific Blue control antibody.

Example 40: Preparation of Polymer-Dye Conjugate

The polymer is conjugated to a dye, Dylight 594, and purified in amanner similar to the methods as described in Example 36. FIG. 23depicts a comparison of the florescence of the dye (DyLight594) andpolymer-dye conjugate. The dye was excited at 594 nm and the polymer-dyeconjugate at 380 nm.

Example 41: Fluorescent Immunoassay (ELISA) with Streptavidin-ConjugatedPolymer

An immunoassay for human IgG was developed as a demonstrative system in96 well plate format. In further embodiments, similar functionalitywould be equally applicable in other formats including suspendedmicrospheres and protein chip microarrays.

Step 1: Preparation of Reagents

Wash concentrate was prepared by dissolving 79.2 g Tris base pre-setcrystals (pH 7.7), 225 g sodium chloride and 0.5 g Thimerosol in 1000 mLdeionised water. Wash solution was prepared by adding 100 mL washconcentrate to 2400 mL deionised water. Subsequently, 10 mL 10% TritonX-100 was added. The basic assay buffer was prepared by dissolving 14.8g Tris base pre-set crystals (pH 7.7), 18 g sodium chloride and 0.5 gThimerosol in 2000 mL Milli-Q water (conductivity 18.2 mΩcm).Subsequently, 2 mL 10% Tween 20 and 10 g Bovine Serum Albumin FractionV, essentially gamma globulin free were added. The solution was filteredand stored at 4° C.

Step 2: Preparation of Capture Antibody Coated Plates

Capture antibody was coated onto the surface of Nunc white Maxisorp 96well plates at a concentration of approximately 1 microgramme per well.The plates were sealed and stored overnight at 4° C. Subsequently, theplates were washed once with wash solution and tapped dry on absorbentpaper. Unless otherwise stated all plate washing in this example wasperformed on an automated microtitre plate washer. Two hundred and fifty(250) microliters of blocking buffer (0.1M PBS containing 2% BSA) wereadded to each well, the plates re-sealed and stored at 4° C. until use.

Step 3: Immunoassay

Capture antibody-coated microtitre plates were washed twice with washsolution and tapped dry on absorbent paper. Two hundred (200) μL ofeither assay standard or experimental unknown sample were added inquadruplicate to appropriate wells of the coated plate. The plates wereincubated on a shaker for 2 hours at 18° C. Subsequently, the plateswere washed three times with wash solution, tapped dry on absorbentpaper, and 200 μL of biotinylated detection antibody at a previouslydetermined optimal concentration (diluted in assay buffer and filteredbefore use) were added to each well. The plates were incubated on anorbital shaker at ambient temperature for a further 60 minutes. Theplates were then washed three times and tapped dry on absorbent paper.Two hundred (200) μL of 0.2 micron syringe filtered Streptavidin-polymerconjugate as prepared in Example 30 diluted to a concentrationpreviously determined as suitable in assay buffer. The polymer was afluorene polymer with neutral PEG11 side chains and an amine conjugationsite. The plates were incubated on an orbital shaker at ambienttemperature for a further 2 hours. The plates were then washed sixtimes, tapped dry, turned around 180°, and re-washed a further sixtimes. The plates were again tapped dry on absorbent paper. Two hundred(200) μL of filtered release reagent (0.1M sodium hydroxide, 2% TritonX-100) were added using a multi-channel pipette, the plates shaken for60 minutes at ambient temperature and the fluorescence measured with aVictor Fluorometer. The plate was then sealed, stored overnight at 4° C.and re-read in the Victor Fluorimeter the following morning.Fluorescence counts were analysed using the Multicalc Software fromPerkin Elmer to determine lower limit of assay detection and assortedsimilar parameters. Alternative conditions were also evaluated torelease the conjugate from the well plate surface to improve thefluorescent readout. A representative data set is shown in FIG. 24.Comparisons were also made to commercially available SA-dye conjugates.The polymer conjugates demonstrated superior detection limits relativeto the dye conjugates as was expected due to the collective opticalproperties.

Example 42: Synthesis, Conjugation and Application of Para-PhenyleneVinylene Co-Polymer with Active Functional Linker for Bioconjugation

Poly(1,4-(di2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yl2,5-dibromoterephthalate)-vinyl-alt-para(2-methoxy-5-2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-ylbenzene)-vinylene) with phenylbutoxyamino termini.

Di-2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yl2,5-dibromoterephthalate (2.0 g, 1.52mmol),34-(4-methoxy-2,5-divinylphenoxy)-2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan(1.11 g, 1.52 mmol), palladium acetate (13.6 mg, 0.061 mmol),tri-o-tolylphosphine (37 mg, 0.121 mmol), triethylamine (1 mL, 7.6 mmol)and 4 mL of DMF were combined in a small round bottom flask, equippedwith a Teflon stribar, fitted with a needle valve and transferred to aSchlenk line. The solution was degassed via three freeze-pump-thawcycles, put under nitrogen and heated to 100 C with constant stirringovernight. Nextdi-2,5,8,11,14,17,20,23,26,29,32-undecaoxatetratriacontan-34-yl2,5-dibromoterephthalate (2.0 g, 1.52 mmol) (100 mg, 5 mol %), palladiumacetate (5 mg), and tri-o-tolylphosphine and 0.5 mL DMF were combined ina small round bottom flask which is fitted with a needle valve andtransferred to the Schlenk line. The solution was degassed via threecycles of freeze-pump-thaw and once warmed to room temperature wastransferred to the polymerization reaction via cannula to exclude airand moisture. Allowed the mixture to react overnight. Next4-(4-bromophenoxy)butan-1-amine (43 mg, 15 mol %) and 0.5 mL of DMF werecombined in a small round bottom flask, equipped with a Teflon stribar,fitted with a needle valve and transferred to a Schlenk line. Oncewarmed to room temperature the solution was transferred to thepolymerization reaction via cannula to exclude air and moisture. Allowedthe mixture to react overnight. The next day the reaction was cooled toroom temperature and the bulk of triethylamine was removed under vacuum.The reaction mixture was diluted with ˜30 mL of water and filteredthrough G 6 glass fiber filter paper. The filtrate was transferred toseveral Amicon filters (10 kDa cutoff) to concentrate the polymer andremove DMF. The remaining water is removed under vacuum and the residueis extracted into methylene chloride. The methylene chloride solution isdried over magnesium sulfate and filtered. The solvent is removedleaving behind a dark red thick oil, approximately 900 mg.

The polymer was found to have a Mn of 20,400 g/mol as determined by GPCanalysis relative to polystyrene standards. Incorporation of the aminelinker was verified by conjugating a dye to the final polymer asdescribed in Example 38.

The polymer was then conjugated to streptavidin protein as follows:Amine polymer was dissolved at 50 mg/ml and desalted and bufferexchanged into 100 mM phosphate buffer pH 7.5. Polymer concentration wasassessed by absorbance and 25 molar equivalents of SMCC (10 mg/ml inanhydrous DMSO) added. The reaction was mixed for 60 mins at roomtemperature and then desalted and buffer exchanged into PBS pH7.0+5 mMEDTA prior to repeat polymer concentration determination andconfirmation of malemide functionality by SAMSA-fluorescein dye test.Streptavidin (5 mg/ml in 100 mM phosphate buffer pH7.5) was activated byaddition of 20 molar equivalents of SATA (5 mg/ml in anhydrous DMSO).The reaction was mixed at room temperature for 60 mins prior toquenching (>15 mins room temp) with 10% (v/v) 50 mM EDTA, 2.5Mhydroxylamine pH7.0. The activated protein was desalted and bufferexchanged into the same buffer as the activated polymer prior to anperformance of an Ellman's assay to confirm and quantify thiolincorporation. Both the activated polymer and streptavidin were used asfollows without delay. A greater than order of magnitude molar excess ofSMCC activated polymer was added to the SATA activated streptavidin andthe two mixed for 2 hours at room temperature prior to quenching with 20molar equivalents of N-ethylmaleimide which was mixed in for 15 minutesat room temperature. Ion exchange and size exclusion chromatography wereused to purify the bioconjugate of unreacted polymer and streptavidin.Appropriate fractions were pooled to maximize yield and performance andthen concentrated by ultrafiltration.

The conjugate was tested and its performance compared to a commerciallyavailable streptavidin-phycoerythrin (SA-PE) conjugate designed forpurpose in a model Luminex xMap assay (FIG. 27, left). Donkey anti-mouseIgG antibody was covalently conjugated to xMap beads. A standard curvetitration of Mouse IgG was then performed under standard Luminex xMapassay conditions (FIG. 27, right). Replicate samples were detected usingeither 4 μg/mL streptavidin-phycoerythrin or streptavidin conjugatedpolymer conjugate prepared as above (concentration not rigorouslycontrolled). Samples were then read on a Luminex instrument. Absolutesignals were found to be lower using the conjugated polymer. This ispartially attributed to a non-ideal match between the polymer spectraand the excitation and emission optics in the instrument as well as theputative lower concentration of detection reagent used compared with thecommercially available phycoerythrin product. However, the proportionalbackground (non specific signal) from the polymer was also markedlylower resulting in a very comparable lower limit of detection for bothdetection formats (Fluorescence highest point in standardcurve/fluorescence zero concentration of analyte (MFI/zero): 21.8 PE,26.6 Polymer).

Example 43: Synthesis of a Fluorene Co-Polymer with a DPP Band GapModifying Unit

To a 25 mL round-bottomed flask were added: PEGylated dibromo-DPPmonomer (110 mmol), PEGylated fluorene diboronicester (110 mmol), THF(2.4 mL) solvent, 2M K₂CO₃ (1.6 three freeze-pump-thaw cycles and thenstirred under argon at 80 C over night. The resulting mixture wasallowed to r.t. and diluted with water. Polymer was collected afterdichloromethane extraction.

The resulting polymer was found to have an absorption maxima at 520 nmand emission maxima at 590 nm with quantum yield of 6% in water. Thepolymer had a MW estimated at 16,000 by GPC analysis relative topolystyrene standards and was soluble in water, methanol anddichloromethane.

End linker incorporation can be performed using methods similar to thosedescribed above and including methods described in Examples 9, 10 and11.

Example 44: Synthesis of a Substituted Divinylbenzene Polymer

Methods used to prepare the polymer above were similar to those providedin Example 38. General methods for the preparation of divinylbenzenepolymers as disclosed herein may be derived from known reactions in thefield as well as methods found herein, and the reactions may be modifiedby the use of appropriate reagents and conditions, as would berecognized by the skilled person, for the introduction of the variousmoieties found in the formulae as provided herein.

Example 45: Conjugation of Polymer to an Amine on a Primary Antibody

Procedure for Production of NHS Ester Polymer-Antibody Conjugate

Primary monoclonal antibody, anti-CD4 (RPA-T4 clone) was desalted, andexchanged into 50 mM NaHCO₃ buffer, pH 8.2 at 1 mg/mL. Enriched NHSfunctionalized polymer was dissolved into anhydrous dimethyl sulfoxide(DMSO) at 100 mg/mL. Polymer solution was added at 30 fold molar excessof antibody into the antibody solution and allowed to mix by agitationfor 3 hours at RT. Protein concentration was adjusted with buffer priorto incubation to ensure the volume of organic solvent was <10% the totalvolume. Following ultrafiltration over a 10KDa MWCO filter device, ionexchange and size exclusion chromatographic techniques were then used topurify the bioconjugate of unreacted polymer and antibody, respectively.Appropriate fractions were pooled to maximize yield and buffer exchangedinto PBS+0.05% NaN₃ and simultaneously concentrated by ultrafiltrationas above. Degree of labeling (indicated as p above) was determined viaabsorbance at 405 nm and a corrected 280 nm value. The polymer conjugate(CJ04-02) provided in Example 39 (FIG. 22) had an F/P (# of polymers perantibody) of approximately 2.04. This conjugate demonstrated flowperformance as determined by stain index measurements which were greaterthan 3 fold higher than a Pacific Blue control conjugate of the sameantibody.

Example 46: Conjugation of Polymer to an Antibody Using Malemide/ThiolChemistry Malemide/Thiol Conjugation of Polymers to Partially ReducedAntibodies

Secondary antibody, goat anti-mouse IgG (H+L) was reconstituted inPBS+10 mM acetic acid and desalted/exchanged into 50 mM Tris-HCl buffer,pH7.4 at 1.0 mg/mL. TCEP (tris(2-carboxyethyl)phosphine) was dissolvedin 50 mM Tris-HCl buffer, pH7.4, added at 6 molar excess with a finalTCEP concentration of 10 mM and mixed for 30 minutes at roomtemperature. The modified protein was purified over a PD-10 desaltingcolumn to remove excess TCEP and exchanged into reaction buffer, 100 mMNaPO4, pH 6.5 reaction buffer with 10 mM EDTA. Amine-activated polymerwas dissolved in anhydrous DMSO at 10 mg/mL and mixed withsuccinimidyl-4-(N-maleimidomethyl)cyclohexane- 1-carboxylate (SMCC)linker. The linker was added at 50 mg/mL, 20 molar excess in DMSO to thepolymer solution and activated by diisopropylethylamine (DIPEA). Thereaction was purified over Amicon Ultra centrifugation filters andexchanged into reaction buffer, 100 mM NaPO4, pH 6.5 reaction bufferwith 10 mM EDTA. Immediately following disulfide reduction, maleimidefunctionalized polymer in reaction buffer at 10 mg/mL was added in 20molar excess of antibody and allowed to mix for 4 hours. Ion exchangeand size exclusion chromatographic techniques were then used to purifythe bioconjugate of unreacted polymer and antibody, respectively. Degreeof labeling (indicated as p above) is determined via absorbance at 405nm and a corrected 280 nm value. The polymer conjugate provided inExample 36 (FIG. 20B) had an F/P (# of polymers per antibody) ofapproximately 2. This conjugate demonstrated flow performance asdetermined by stain index measurements which were greater than 4 foldhigher than a Pacific Blue control conjugate of the same antibody.

Malemide/Thiol Conjugation of Polymers to Thiol Modified Antibodies

Secondary antibody, goat anti-mouse IgG (H+L) was reconstituted inPBS+10 mM acetic acid and desalted/exchanged into 100 mM phosphate pH7.5buffer. SATA (N-succinimidyl-S-acetylthioacetate) was dissolvedanhydrous DMSO, added at 15 molar excess and mixed for 60 minutes atroom temperature. After quenching with a hydroxylamine solution, themodified protein was desalted over a PD-10 column to remove excess SATAand exchanged into reaction buffer, 5 mM EDTA, PBS pH 7.0 buffer.Amine-activated polymer was dissolved in anhydrous DMSO at 10 mg/mL andmixed with succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(SMCC) linker. The linker was added at 50 mg/mL, 20 molar excess in DMSOto the polymer solution and activated by diisopropylethylamine (DIPEA).The reaction was purified over Amicon Ultra centrifugation filters andexchanged into reaction buffer, 5 mM EDTA, PBS pH 7.0 buffer.Immediately following activation of the antibody, maleimidefunctionalized polymer in reaction buffer at 10 mg/mL was added in 20molar excess of antibody and allowed to mix for 4 hours. Ion exchangeand size exclusion chromatographic techniques were then used to purifythe bioconjugate of unreacted polymer and antibody, respectively. Degreeof labeling (indicated as p above) is determined by absorbance at 405 nmand a corrected 280 nm value. The resulting purified conjugates wereflow tested in similar fashion as those described in Example 36 for theconjugates prepared using TCEP reduction (data not provided).

The polymer structures defined in Example 39 were used to prepareprimary antiCD4 (RPA-T4) antibody conjugates in similar fashion to thosedescribed in the example above. 30 eq of polymer were reacted with theSATA modified antibody (CJ11-2, FIG. 22) and TCEP reduced antibody(CJ13-1, FIG. 20D and FIG. 22) to produce polymer conjugates for testingin flow cytometry assays after purification. SMCC modified polymers fromExamples 23 and 26 were also successfully conjugated to antiCD4 (RPA-T4)and antiCD8 (RPA-T8) antibodies using the TCEP reduction method. DTTreduction was also successfully performed in place of TCEP. Performancein flow cytometry of the antiCD4 and antiCD8 conjugates were evaluatedin similar fashion to those described in Example 39 (FIG. 22).

Example 47: Polymer Conjugation to a DNA Oligomer

Azide Polymer Synthesis for Click Conjugation to Alkyne Terminated DNAOligo

A solution of azidohexanoic acid NHS ester (2.5 mg) in anhydrous DMF(100 μL) was added to a solution of the amine-functional polymer (9.9mg) in anhydrous DMF (100 μL) under argon. Diisopropylethylamine (2 μL)was then added. The reaction was agitated at room temperature for 15hours. Water was then added (0.8 mL) and the azide-modified polymer waspurified over a NAP-10 column. The eluent was freeze dried overnight.Yield 9.4 mg, 95%.

Oligo Synthesis with pendant alkyne (hexyne) for Click conjugation toAzide polymer

The 3′ propanol oligo A8885 (sequence YATTTTACCCTCTGAAGGCTCCP, whereY=hexynyl group and P=propanol group) was synthesized using 3′ spacerSynBase™ CPG 1000 column on an Applied Biosystems 394 automated DNA/RNAsynthesizer. A standard 1.0 μmole phosphoramidite cycle ofacid-catalyzed detritylation, coupling, capping and iodine oxidation wasused. The coupling time for the standards monomers was 40 s, and thecoupling time for the 5′ alkyne monomer was 10 min.

The oligo was cleaved from the solid support and deprotected by exposureto concentrated aqueous ammonia for 60 min at room temperature, followedby heating in a sealed tube for 5 h at 55° C. The oligo was thenpurified by RP-HPLC under standard conditions. Yield 34 OD.

Solution Phase Click Conjugation: Probe Synthesis

A solution of degassed copper sulphate pentahydrate (0.063 mg) inaqueous sodium chloride (0.2 M, 2.5 μL) was added to a degassed solutionof tris-benzo triazole ligand (0.5 mg) and sodium ascorbate (0.5 mg) inaqueous sodium chloride (0.2 M, 12.5 μL). Subsequently, a degassedsolution of oligo A8885 (50 nmole) in aqueous sodium chloride (0.2 M, 30μL) and a degassed solution of azide polymer (4.5 mg) in anhydrous DMF(50 μL) were added, respectively. The reaction was degassed once morewith argon for 30 s prior to sealing the tube and incubating at 55° C.for 2 h. Water (0.9 mL) was then added and the modified oligo waspurified over a NAP-10 column. The eluent was freeze-dried overnight.The conjugate was isolated as a distinct band using PAGE purificationand characterized by mass spectrometry. Yield estimated at 10-20%.

Fluorescence Studies

The oligo-polymer conjugate was used as a probe in fluorescence studies.The probe was hybridized with the target A8090 (sequenceGGAGCCTTCAGAGGGTAAAAT-Dabcyl (SEQ ID NO: 2)), which was labeled withdabcyl at the 3′ end to act as a fluorescence quencher. The target andprobe were hybridized, and fluorescence monitored in aPeltier-controlled variable temperature fluorimeter. The fluorescencewas scanned every 5° C. over a temperature range of 30 C to 80° C. at arate of 2° C./min. FIG. 25 shows increasing fluorescence intensity oremission with increasing temperature, indicating that as theprobe-target pair melt, the polymer and quencher separate andfluorescence is recovered.

Polymer conjugation to nucleic acids can also be performed using methodsadapted from the protocols described in Examples 14, 45 and 46.

Example 48: Purification of Polymer Antibody Conjugates

Polymer antibody conjugates produced via the protocols described inExamples 45, 46 and 49 were purified using a two step method. First ionexchange is used to remove free, unreacted polymer. As the polymersdescribed in this invention do not possess any formal charge they do notbind to the ion exchange media. Proteins (antibodies), however, docontain charged groups and are commonly bound to various ion exchangemedia for purification. In the examples provided the pH and conductivityof the conjugate solution (post reaction) was lowered to improve thebinding of the free antibody and conjugate to the cationic exchangeresin. After loading the conjugate, the resin is washed to baseline(measuring both 280 and 407 nm absorbance) to ensure all free polymer isremoved. Bound antibody and polymer antibody conjugate are eluted byincreasing the pH and ionic strength. A representative example of thisseparation is provided below in FIG. 26 (left) where the left peakrepresents the free polymer and the right peak the eluted conjugate andfree protein. Removal of free polymer can also be achieved usingaffinity chromatograph methods in a similar fashion. Specific affinityresin can be used to bind the free protein and conjugate while removingpolymer.

After the polymer is removed, the conjugate solution is concentrated andloaded on a size exclusion column to separate any un-reacted or freeantibody from the polymer. The polymer compositions described inExamples 43 and 44 elute much earlier than then antibodies despitehaving a lower molecular weight. This is expected to be a result of therigid polymer structure. The conjugates thus elute well before any freeantibody providing near base line separation of the desired conjugate.Isolating fractions near the center of the distribution also ensures nofree antibody is included. A representative example of this separationis provided below in FIG. 26 (right) where the left peak represents theconjugate and the small peak on the far right the free antibody.Retention times of the individual components was verified in anindependent experiment.

Taken together the purification ensures that both free antibody and freepolymer are removed. Purity of the resulting conjugates is reasonablyestimated at >95%. Pooled samples can be concentrated and concentrationmeasured by absorbance at 280 and 407 nm, being sure to correct for thepolymer absorbance at 280 nm. Such measurements also allow for thedetermination of polymer to antibody labelling ratios (F/P).

Example 49: Dye Labeling and Linker Activation of Tandem Polymer

Tandem Dye Conjugation

In a glovebox, 93 mg tandem polymer (from Example 26) was dissolved at15 mg/mL in anhydrous DMSO in a glass vial with stir bar. 22.5 mgCy3-NHS ester was also dissolved at 15 mg/mL in anhydrous DMSO and addedto the polymer solution, followed by 0.3 mL diisopropylethylamine. Afterstirring for 48 h at room temperature, the solution was diluted to 90 mLwith 20% EtOH in water and concentrated over Amicon Ultra-15 filters.The retentate was repeatedly diluted and concentrated over the filtersuntil excess Cy3 was removed. 90% yield. Labeling and linker contentwere validated by measuring and taking the ratio of polymer and dyeabsorbance as described in Example 38.

Amine Modification of Tandem (Aqueous Conditions)

100 mg of polymer-dye conjugate was dissolved at 150 mg/mL in ethanol.This was added dropwise to 6 mL 50 mM MES buffer (pH 5) at 4° C. 38 mgN-hydroxy succinimide was added in one portion, and the solution wasstirred to dissolve the solids. After dissolution, 192 mg ofN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride was addedin portions while stirring. After stirring the solution for 30 minutes,33 μL of ethylene diamine was added. After stirring overnight at roomtemperature, the solution was diluted to 90 mL with 20% EtOH in waterand concentrated over Amicon Ultra-15 filters. The retentate wasrepeatedly diluted and concentrated over the filters a total of fourtimes to remove impurities. 90% yield, 60% conversion. Linker conversionwas verified by conjugating a second dye to the terminal amine asdescribed in Example 38.

Tandem Conjugation to a Primary Antibody

Primary monoclonal antibody, anti-CD8 (RPA-T8 clone) wasdesalted/exchanged into 5 mM EDTA, 50 mM phosphate 150 mM NaCl pH 7.0buffer. TCEP (tris(2-carboxyethyl)phosphine) was dissolved water andadded at 12 molar excess and mixed for 90 minutes at 30° C. The modifiedprotein was purified over a PD-10 desalting column to remove excess TCEPand exchanged into 5 mM EDTA, 50 mM phosphate 150 mM NaCl pH 7.0 buffer.

Amine-activated tandem polymer was dissolved in ethanol at 50 mg/mL andthis solution was mixed with two volumes of 100 mM phosphate pH 7.5buffer. This solution was then desalted/exchanged into 100 mM phosphatepH 7.5 buffer using a PD-10 desalting column. To this solution was added25 molar excess ofsuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC)linker (prepared as a 10 mg/ml solution in anhydrous DMSO). Theresulting solution was rollermixed at 20° C. for 60 minutes before beingdesalted/exchanged into 5 mM EDTA, 50 mM phosphate 150 mM NaCl pH 7.0buffer using a PD-10 desalting column. Immediately following disulfidereduction, the maleimide functionalized polymer was added in 25 molarexcess of antibody and allowed to mix for 2 hours at 20° C. Ion exchangeand size exclusion chromatographic techniques were then used to purifythe bioconjugate of unreacted polymer and antibody, respectively. Degreeof labeling (indicated as p below) is determined via absorbance and acorrected 280 nm value.

Flow Cytometry Analysis of Polymer Tandem Conjugate in MulticolorExperiment

The resulting antiCD8 Tandem conjugate was evaluated on bothcompensation beads and whole blood samples on a flow cytometer. Antimouse IgG compensation beads were used to capture the antibody andquantify signal spill over into detection channels (detectors withunique emission filters) other than that intended for the conjugate.FIG. 28 (left) shows the signal measured when the tandem conjugate wasexcited with a violet laser with emission detected using a 610 nm filtermatched to the conjugates emission (labeled QDotA). Crosstalk into theflow cytometer's other channels paired with the violet excitation laser(DAPI-A and AmCyan-A) and two channels off the 488 nm laser (FITC-A andPE-A) are also shown in this panel of the figure. The data show minimalcrosstalk in the 450/50 nm filter (DAPI-A) which specifically detectsresidual polymer (donor) emission. The significantly higher signal fromthe Cy3 reporter on the Tandem (610 nm filter) relative to the otherchannels above illustrates that minimal compensation (maximally no morethan 6% in this example and case by case often much lower) is required.

The Tandem anti CD8 conjugate was subsequently evaluated in a 4 colorflow assay with other labeled antibodies (anti CD3 Pacific Blue, antiCD45 Phycoerythrin and anti CD4 fluorescein) on whole human blood from ahealthy volunteer using staining and analysis protocols in accord anddeveloped from Example 39. The data in FIG. 28 (right) clearly show thecompatibility of the Tandem label with common multicolour flow cytometryinstrumentation, reagents and protocols. Specifically, intense andspecific staining of CD8 positive lymphocytes is observed and within theCD4 positive subset ready discrimination of CD8 expressive and nonexpressive cells is clear

Collectively the data highlight the viability of the polymer-dye Tandemconjugates in multicolor flow assays as described in the disclosedinvention (See, e.g., FIG. 20 and FIG. 22).

Example 50: Validation of Non-Ionic Polymer Side Chains for WaterSolubility and Flow Cytometry Application

A series of different polyfluorene polymers were produced to investigatethe interaction of water soluble conjugated polymers with cells. Thiswas done by first synthesizing a range of monomers substituted withdifferent solublizing side chains (e.g., PEG-, sulfonate-, quaternaryamine-, zwitterion-substituted) which were then polymerized using Suzukicoupling. The purpose was to determine what influence the side chainshad on both non-specific cell binding and polymer solubility in typicalbuffers used in biological assays, particularly those used in flowcytometry (e.g. PBS and DPBS).

The number and property diversity of polymer candidates synthesized madeit impractical to produce purified conjugates of each for flow cytometrytesting. Thus, a system was developed to score each candidate polymerbased on its contribution to non-specific binding to cells. Such asystem enabled ranking of polymers, with predictive value on whetherthey would perform sufficiently once conjugated. A Non-specific Binding(NSB) “Index” was developed around a Jurkat cell model (lymphocyte cellline). In this, cells were incubated with a fixed concentration of eachpolymer, washed, and analyzed by flow. FIG. 33 displays the outcomefollowing such analysis, and illustrates the wide variation in signalgenerated by each polymer type. The polymers in FIG. 33 were evaluatedwith a phthalamide protecting group on the pendant amine with theexception of P9.

The data ranks these polymers in terms of signal generated purely byNSB. More accurate assessment of relative NSB was enabled by adjustingfurther normalizing the flow signal by differences in fluorescenceefficiency (crude assessment of quantum yield) of each form of polymerwhen assayed independently in stain buffer using 405 nm excitation on afluorometer and monitoring emission in the range of 420-460 nm (toestimate a 440/40 nm filter in the cytometer). Representative polymersP5, P2, P9 and P12 showed increasing NSB relative to unstained cells(far left curve, intensity represented on x-axis).

The data in FIG. 34 go on to highlight the difference in polymersproduced with neutral, non-ionic PEG side chains (designated P20) versesthose which also incorporate anionic side chains (designated P4). Thedata are histograms collected from flow cytometry analysis (405 nmexcitation in a BD LSR-II cytometer) using a Jurkat cell line as in FIG.33. The panel on the left shows unstained cells and a negative control(cells treated with a non-specific Pacific Blue labeled conjugate) whichare the two curves on the far left. Little if any non-specific stainingis observed for the Pacific Blue control. In this same panel, however,curve on the right represents cells treated with the anionic P4 polymerand has a clear off set in signal (x-axis) as shown. Conversely theneutral polymer P20 showed almost no off set from the untreated cellswhich is in line with the Pacific Blue control. The panel in the middlerepresents a range of different polymer and polymer side chaincombinations tested on the same cells.

The data highlighted the advantage of neutral side chains. Thisadvantage has also translated to other assay formats including platebased immunoassays and cytometric bead arrays (data not shown). Theneutral side chains also unexpectedly resulted in a significant increasein the solubility of the conjugated polymers in aqueous solutionsrelative to those made previously with ionic side chains. This wasparticularly true in buffers containing even moderate ionic strength(such as those used in basic cell protocols). The solution quantumyields were also seen to increase, possibly due to the higher aqueoussolubility (and less aggregation). The poor solubility in buffers alsomade protein conjugation more difficult and streptavidin conjugatesproduced from P4 showed signs of aggregation in typical assay bufferssuch as phosphate buffered saline (PBS). This was not true of polymersand conjugates produced in other examples disclosed herein.

Example 51: Purification and Characterization of Polymer-AvidinConjugates Gel Analysis of Polymer-Avidin Conjugates

To verify successful conjugation to avidin (AvDN), an agarose gelelectrophoresis method was developed and used to assess the relativemobility of AvDN as a function of the degree of conjugation with polymer(FIG. 35). Prior to gel loading, the conjugation reaction was stainedwith biotinyl-fluorescein, which bound polymer-AvDN conjugate and freeAvDN. Electrophoresis was performed in 0.8% agarose gels, poured and runin a buffer of 10 mM Sodium Borate, pH 11. The gel was visualized underUV illumination (to visualize the polymer) and by 532 nm excitation (tovisualize fluorescein) to assess the degree of conjugation. Under UVillumination, a single band was observed for polymer. Under 532 nmexcitation, bands were observed for unbound biotinyl-fluorescein,unreacted AvDN, and polymer-AvDN conjugate which coincided with the freepolymer band, indicating that unreacted polymer co-eluted withpolymer-AvDN conjugate (FIG. 35). Conjugation was confirmed by theintensity of the conjugate band.

The key at the top of the gel images (FIG. 35) indicates whichcomponents were included in the conjugation reaction, as well as whetherthe samples were pre-incubated with biotinyl fluorescein prior toloading and electrophoresis. The image on left visualizes polymer byUV-excitation, whereas the image on right captures the result offluorescein excitation. On the right image, biotinylated fluorescein canbe seen associating with polymer when conjugation was performed in thepresence, but not in the absence, of hetero-bifunctionalNHS-ethoxy-maleimide linkers (linkers were used to functionalize thepolymer amine, while protein amines were partially converted to thiolsusing Traut's reagent, prior to the maleimide-thiol coupling).Abbreviations: AvDN=avidin DN, AA1=polymer, Linker=hetero-bifunctionalNHS-Maleimide linker included in the reaction, Biot-F=biotinylfluorescein pre-staining before electrophoresis.

Purification: Removal of Unreacted Avidin by SEC Chromatography

The crude conjugate mixture was fractionated on a Superdex 200 sizeexclusion column, while fractions were monitored by UV absorbance (FIG.36, top). To validate the method, fractions were analyzed by agarose gelelectrophoresis. As described above, this method of electrophoresis madeit possible to visualize the degree to which avidin was attached topolymer, and in this case to analyze the composition of each fractionfrom the column. Selected fractions were incubated withbiotinyl-fluorescein (1 molar equivalent relative to avidin) prior togel loading, with biotinyl-fluorescein loaded separately as a marker(leftmost lane, FIG. 36, bottom). Electrophoresis was performed in 0.8%agarose gels, poured and run in a buffer of 10 mM Sodium Borate, pH 11.The gel was visualized by 532 nm excitation. Retardation offluorescein-visualized bands for fractions C2-C6 indicates purifiedpolymer-avidin conjugate, while the two bands observed for fraction C8indicate a mixture of polymer-avidin conjugate and free avidin.Fractions C10-D2 show only free avidin.

Evaluation of Conjugation Efficiency by Gel Analysis

In order to determine the best ratio of polymer to streptavidin inconjugation reactions, the molar equivalents of polymer to streptavidinwere varied from 0-24 equivalents. Post conjugation, the conjugationproducts were incubated with biotinyl-fluorescein prior toelectrophoresis. The gel was visualized by UV illumination and 532 nmexcitation (FIG. 37). At 0 molar equivalents of polymer to streptavidin,free streptavidin is observed as a band with relatively high mobility.As the molar equivalents for polymer are increased from 3 equivalents to12 equivalents, the free streptavidin band decreases in intensity whilethe polymer-streptavidin conjugate band increases in intensity. At 24equivalents of polymer, only the conjugate band is observed by 532 nmexcitation.

Impact of Purification on Conjugate Performance on Cell Analysis by FlowCytometry

Purification of polymer streptavidin conjugates (polymer structureexemplified in Example 9, denoted P30 in FIG. 38) was performed todetermine the impact on flow cytometry performance. Cation-exchangechromatography was implemented in purification to improve removal ofexcess free polymer. Uncharged polymer eluted in the flow-through whileprotonated amines on proteins were retained by the media. Thus,streptavidin, whether conjugated to polymer or unreacted, was retained.This ion exchange phase of purification was kept simple with a stepgradient, which resulted in co-elution of conjugated and unreacted SA.Further fractionation was enabled by subsequent size-exclusionchromatography, which provided better resolution of conjugate from freeSA. Performance benefits in flow cytometry of this new purificationmethod were observed using Jurkat cells incubated withpolymer-streptavidin conjugate which were analyzed by flow cytometry.Comparisons were made between crude samples (FIG. 38—top) and purifiedconjugates (FIG. 38 bottom). Commercially available PacificBlue-streptavidin conjugates were used as a comparator for brightness,nonspecific binding, and stain index. An improvement in overall StainIndex of approximately 3-fold was shown for Jurkat cells, with similarNSB for both Polymer conjugates and PB-SA based on the histograms shownin FIG. 38. Testing in blood (data not shown) indicated a significantreduction in NSB to levels similar to PB-SA upon conjugate purification.

In a separate experiment with a similar polymer (exemplified in Example11), conjugates with varying polymer to streptavidin ratios wereobtained by SEC. Those with the higher ratio provided flow performancerelative to those with lower labeling. Ratios were determined based on aratio of absorbance at 385 nm/280 nm. Relative performance to a PacificBlue control showed an increase from 10.9 times higher stain index(385/280 ratio of 3.6) to a stain index 13.8 times that of Pacific Blue(A385/280 ratio of 4.7).

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A polymer-biomolecule conjugate comprising: awater soluble conjugated polymer; and a biomolecule covalently linked tothe water soluble conjugated polymer; wherein the water solubleconjugated polymer is of the formula:

wherein: each R is independently a non-ionic side group capable ofimparting solubility in water; MU is a polymer modifying unit or bandgap modifying unit that is evenly or randomly distributed along thepolymer main chain and is optionally substituted with one or moreoptionally substituted substituents selected from halogen, hydroxyl,C₁-C₁₂ alkyl, C₂-C₁₂ alkene, C₂-C₁₂ alkyne, C₃-C₁₂ cycloalkyl, C₁-C₁₂haloalkyl, C₁-C₁₂ alkoxy, C₂-C₁₈ (hetero)aryloxy, C₂-C₁₈(hetero)arylamino, a C₂-C₁₈ (hetero)aryl group and(CH₂)_(x′)(OCH₂CH₂)_(y′)OCH₃ where x′ is independently an integer from0-20 and y′ is independently an integer from 0 to 50; L₁ and L₂ arelinkers each independently an aryl or a heteroaryl group evenly orrandomly distributed along the polymer main chain and are substitutedwith one or more pendant chains terminated with: i) a functional groupselected from amine, carbamate, carboxylic acid, carboxylate, maleimide,activated ester, N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone,azide, alkyne, aldehyde, thiol, and protected groups thereof forconjugation to a molecule or biomolecule; or ii) a conjugated organicdye or the covalently linked biomolecule; G₁ and G₂ are eachindependently selected from hydrogen, halogen, alkyne, optionallysubstituted aryl, optionally substituted heteroaryl, halogen substitutedaryl, boronic acid substituted aryl, boronic ester substituted aryl,boronic ester, boronic acid, optionally substituted fluorene and aryl orheteroaryl substituted with one or more pendant chains terminated with:i) a functional group selected from amine, carbamate, carboxylic acid,carboxylate, maleimide, activated esters, N-hydroxysuccinimidyl,hydrazine, hydrazide, hydrazone, azide, alkyne, aldehyde, thiol, andprotected groups thereof for conjugation to a molecule or biomolecule;or ii) a conjugated organic dye or the covalently linked biomolecule; nis an integer from 1 to about 10,000; and a, b, c and d define the mol %of each unit within the structure which each can be evenly or randomlyrepeated and where a is a mol % from 10 to 100%, b is a mol % from 0 to90%, and each c and d are mol % from 0 to 25%.
 2. Thepolymer-biomolecule conjugate according to claim 1, wherein thebiomolecule is a sensor biomolecule capable of interacting with a targetmolecule.
 3. The polymer-biomolecule conjugate according to claim 1,wherein the biomolecule is a protein, a peptide, an affinity ligand, anantibody, an antibody fragment, a sugar, a lipid, a nucleic acid, anaptamer, avidin, streptavidin, neutravidin, avidinDN or avidinD.
 4. Thepolymer-biomolecule conjugate according to claim 3, wherein thebiomolecule is an antibody or antibody fragment.
 5. Thepolymer-biomolecule conjugate according to claim 1, wherein MU isselected from optionally substituted benzothiadiazole, optionallysubstituted benzoxidazole, optionally substituted benzoselenadiazole,optionally substituted benzotellurodiazole, optionally substitutednaphthoselenadiazole, optionally substituted4,7-di(thien-2-yl)-2,1,3-benzothiadiazole, squaraine dyes, optionallysubstituted quinoxaline, optionally substituted perylene, optionallysubstituted perylene diimide, optionally substituteddiketopyrrolopyrrole, thienopyrazine low bandgap commercial dyes, olefinand cyano-substituted olefins and isomers thereof.
 6. Thepolymer-biomolecule conjugate according to claim 5, wherein MU isselected from the group consisting of a′-k′ having the structures:

wherein * is a site for covalent attachment to the backbone of theconjugated polymer and each R is a side group capable of impartingsolubility in water.
 7. The polymer-biomolecule conjugate according toclaim 6, wherein MU is selected from one of the following:

wherein * is a site for covalent attachment to the backbone of theconjugated polymer.
 8. The polymer-biomolecule conjugate according toclaim 1, wherein MU is a phenyl.
 9. The polymer-biomolecule conjugateaccording to claim 8, wherein MU is

wherein * is a site for covalent attachment to the backbone of theconjugated polymer.
 10. The method according to claim 1, wherein each Ris a non-ionic side group capable of imparting solubility in water inexcess of 10 mg/mL.
 11. The polymer-biomolecule conjugate according toclaim 1, wherein each R comprises an ethylene glycol oligomer.
 12. Thepolymer-biomolecule conjugate according to claim 11, wherein each R is anon-ionic side group comprising mPEG5, mPEG8, mPEG11 or mPEG24.
 13. Thepolymer-biomolecule conjugate according to claim 11, wherein each R isindependently selected from (CH₂)_(x′)(OCH₂CH₂)_(y′)OCH₃ wherein x′ isindependently an integer from 0-20 and y′ is independently an integerfrom 0 to 50, and a benzyl substituted with one or more groups selectedfrom halogen, hydroxyl, C₁-C₁₂ alkoxy and (OCH₂CH₂)_(z)OCH₃ wherein z isindependently an integer from 0 to
 50. 14. The polymer-biomoleculeconjugate according to claim 11, wherein each R is(CH₂)_(x′)(OCH₂CH₂)_(y′)OCH₃ wherein x′ is independently an integer from0-20 and y′ is independently an integer from 0 to
 50. 15. Thepolymer-biomolecule conjugate according to claim 1, wherein each R is adendrimer of PAMAM, PEA, PEHAM, PPI, tri-branched benzoate, or glycerolwith a generation of 1 to 4 and optional terminal substitutions selectedfrom ( - - - - - )CH₂CH₂O)_(j)CH₃ and ( - - - - - )OCH₂CH₂)_(j)CH₃ wherej is an integer from 0 to 25 and the dotted lines ( - - - - - ) are eachindependently selected from any one or a combination of, C₁-C₁₂ alkyl,C₁-C₁₂ alkoxy, C₂-C₁₂ alkene, amido, amino, aryl,(CH₂)_(r)(OCH₂CH₂)_(s)(CH₂)_(r) where each r is independently an integerfrom 0-20, s is independently an integer from 0 to 50, carbamate,carboxylate, C₃-C₁₂ cycloalkyl, imido, phenoxy and C₄-C₁₈(hetero)arylgroup.
 16. The method according to claim 1, wherein wherein L₁ and/or L₂are present and have the structure:

wherein: * is a site for covalent attachment to the backbone of theconjugated polymer; each R′ is different from R and is independentlyselected from H, halogen, C₁-C₁₂ alkyl, (C₁-C₁₂ alkyl)NH₂, C₂-C₁₂alkene, C₂-C₁₂ alkyne, C₃-C₁₂ cycloalkyl, C₁-C₁₂ haloalkyl,C₂-C₁₈(hetero)aryl, C₂-C₁₈(hetero)arylamino, —[CH₂—CH₂]_(r′)—Z¹— whereinZ¹ is OH or —COOH and r′ is an integer from 1 to 20, (C₁-C₁₂)alkoxy-X¹wherein X¹ is —NH₂, —NHCOOH, —NHCOOC(CH₃)₃,—NHCO(C₃-C₁₂)cycloalkyl(C₁-C₄)alkyl-N-maleimide or—NHCO[CH₂—CH₂—O]_(s′)(CH₂)_(s′)NH₂ and each s′ is independently aninteger from 1 to 20, (CH₂)₃(OCH₂CH₂)_(x″)OCH₃ wherein x″ is an integerfrom 0 to 50, and a benzyl optionally substituted with one or morehalogen, hydroxyl, C₁-C₁₂ alkoxy or (OCH₂CH₂)_(y″)OCH₃ wherein y″ is aninteger from 0 to
 50. 17. The polymer-biomolecule conjugate according toclaim 1, wherein L₁ and/or L₂ are present and have the structure:

wherein: * is a site for covalent attachment to the backbone of theconjugated polymer; each R₂₅ is independently a bond, C₁-C₂₀ alkyl,C₁-C₂₀ alkoxy, C₂-C₂₀ alkene, C₂-C₂₀ alkyne, C₃-C₂₀ cycloalkyl, C₁-C₂₀haloalkyl; (CH₂)_(x)(OCH₂CH₂)_(p)(CH₂)_(x) where each x is independentlyan integer from 0-20, p is independently an integer from 0 to 50; aryl,C₂-C₁₈(hetero)aryl, phenoxy, amido, amino, carbamate, carboxylate,carbonates, sulfide, disulfide, or imido groups; and at least one R₂₅ isterminated with a functional group selected from amine, carbamate,carboxylate, carboxylic acid, maleimide, activated ester,N-hydroxysuccinimidyl, hydrazine, hydrazide, hydrazone, azide, alkyne,aldehyde and thiols, or a conjugated organic dye or the covalentlylinked biomolecule.
 18. The polymer-biomolecule conjugate according toclaim 1, wherein L₁ and/or L₂ is conjugated to a signaling chromophore.19. The polymer-biomolecule conjugate according to claim 1, wherein G₁or G₂ comprise the covalently linked biomolecule.
 20. Thepolymer-biomolecule conjugate according to claim 15, wherein at leastone of G₁ and G₂ has the structure:

wherein: * is a site for covalent attachment to the backbone of theconjugated polymer; and R¹¹ is a bond, C₁-C₂₀ alkyl, C₁-C₂₀ alkoxy,C₂-C₂₀ alkene, C₂-C₂₀ alkyne, C₃-C₂₀ cycloalkyl, C₁-C₂₀ haloalkyl,(CH₂)_(x)(OCH₂CH₂)_(p)(CH₂)_(x) wherein each x is independently aninteger from 0-20 and p is an integer from 0 to 50, aryl,C₂-C₁₈(hetero)aryl, phenoxy, amido, amino, carbamate, carboxylate,carbonate, sulfide, disulfide, or imido groups; and is terminated with afunctional group covalently linked to the biomolecule.